Category Archives: Massachusetts Stem Cells

Scientists at Harvard-affiliated Massachusetts General Hospital (MGH) have identified a previously unknown biological pathway that promotes chronic inflammation and may help explain why sedentary people have an increased risk for heart disease and strokes.

In a study to be published in the November issue ofNature Medicine, MGH scientists and colleagues at several other institutions found that regular exercise blocks this pathway. This discovery could aid the development of new therapies to prevent cardiovascular disease.

Regular exercise protects the cardiovascular system by reducing risk factors such as cholesterol and blood pressure. But we believe there are certain risk factors for cardiovascular disease that are not fully understood, said Matthias Nahrendorf of the Center for Systems Biology at MGH. In particular, Nahrendorf and his team wanted to better understand the role of chronic inflammation, which contributes to the formation of artery-clogging blockages called plaques.

Nahrendorf and colleagues examined how physical activity affects the activity of bone marrow, specifically hematopoietic stem and progenitor cells (HSPCs). HSPCs can turn into any type of blood cell, including white blood cells called leukocytes, which promote inflammation. The body needs leukocytes to defend against infection and remove foreign bodies.

When these [white blood] cells become overzealous, they start inflammation in places where they shouldnt, including the walls of arteries.

Matthias Nahrendorf

But when these cells become overzealous, they start inflammation in places where they shouldnt, including the walls of arteries, said Nahrendorf.

Nahrendorf and his colleagues studied a group of laboratory mice that were housed in cages with treadmills. Some of the mice ran as much as six miles a night on the spinning wheels. Mice in a second group were housed in cages without treadmills. After six weeks, the running mice had significantly reduced HSPC activity and lower levels of inflammatory leukocytes than the mice that simply sat around their cages all day.

Nahrendorf explains that exercising caused the mice to produce less leptin, a hormone made by fat tissue that helps control appetite, but also signaled HSPCs to become more active and increase production of leukocytes. In two large studies, the team detected high levels of leptin and leukocytes in sedentary humans who have cardiovascular disease linked to chronic inflammation.

This study identifies a new molecular connection between exercise and inflammation that takes place in the bone marrow and highlights a previously unappreciated role of leptin in exercise-mediated cardiovascular protection, said Michelle Olive, program officer at the National Heart, Lung, and Blood Institute Division of Cardiovascular Sciences. This work adds a new piece to the puzzle of how sedentary lifestyles affect cardiovascular health and underscores the importance of following physical-activity guidelines.

Reassuringly, the study found that lowering leukocyte levels by exercising didnt make the running mice vulnerable to infection. This study underscores the importance of regular physical activity, but further focus on how exercise dampens inflammation could lead to novel strategies for preventing heart attacks and strokes. We hope this research will give rise to new therapeutics that approach cardiovascular disease from a completely new angle, said Nahrendorf.

The primary authors of theNature Medicinepaper are Nahrendorf, who is also a professor of radiology at Harvard Medical School; Vanessa Frodermann, a former postdoctoral fellow at MGH who is now a senior scientist at Novo Nordisk; David Rohde, a research fellow in the Department of Radiology at MGH; and Filip K. Swirski, an investigator in the Department of Radiology at MGH.

The work was funded bygrantsHL142494 andHL139598from the National Heart, Lung, and Blood Institute (NHLBI), part of the National Institutes of Health.

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Exercise found to block chronic inflammation in mice - Harvard Gazette

Its a common refrain in dementia studies: We should have treated them earlier. Its why treating neurodegenerative diseases has been so difficult, Gerhard Koenig, Ph.D., told FierceBiotech.

We believe that the challenges in treating neurodegeneration stem from targeting disease pathologies rather than root causes such as lysosomal dysfunction, as well as from treating patients too late in their disease when significant damage has already occurred, said Koenig, CEO and co-founder of Arkuda Therapeutics, in a statement.

Arkuda is trying to change that. Armed with $44 million from the likes of Atlas Venture and Pfizer Ventures, the Cambridge, Massachusetts-based biotech is going after progranulin, a protein that plays a role in neuronal health and the function of lysosomesorganelles involved in removing waste from cells. Its first target? GRN-related frontotemporal dementia (FTD-GRN), an inherited form of dementia.

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Its starting with FTD-GRN because its an autosomal dominant disease, meaning everyone who has a faulty copy of the GRN gene will have low levels of progranulin and develop dementia, typically in their 40s or 50s. Here, progranulin is both the biomarker and the targetArkuda is using progranulin levels to identify patients who have FTD-GRN and developing small molecules to boost those levels and return lysosomes and neurons to health.

RELATED: Finding of dementia genes could speed drug discovery in Alzheimer's and other brain disorders

Arkuda has shown the approach works in preclinical models and hopes to be testing it in humans in 2021, Chief Business Officer Andy Hu told FierceBiotech.

The series A is designed to enable us to take our lead program all the way through phase 1b proof of mechanism in humans Were also doing work on better understanding of the biology around progranulin, granulin [its subunits] and, more broadly, in the lysosome and we expect to yield some other interesting directions we can take future programs, Hu said. Were also thinking about other potential clinical applications for our lead program.

The company kept mum on what those other applications may be, but Koenig said it has very concrete ideas and internal data to point it toward its next programs. These could include Alzheimers disease, in which the bulk of research has targeted the buildup of amyloid and tau proteins in the brain.

When asked why Arkuda chose FTD-GRN rather than something like Alzheimers disease for its first focus, Koenig emphasized the importance of discipline.

RELATED: Small Alzheimer's hopeful biotech nabs $15M series B for metabolic drug approach

We can have big dreams of what we want to change, but we have to be more precise inour initial approach We need to be disciplined about what we know, which mechanism we are addressing and then later on, look at additional things, he said.

We often talk in these areas about gain of toxic protein function. That is now what we are treating here, Koenig said. We are treating the loss of a protein function I personally believe its a big difference than going after reducing something that is already deposited. Its a differentiated approach, trying to intervene early before the organ is failing and its too late.

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Most cancers kill because tumor cells spread beyond the primary site to invade other organs. Now, a USC study of brain-invading breast cancer cells circulating in the blood reveals they have a molecular signature indicating specific organ preferences.

The findings, which appear in Cancer Discovery, help explain how tumor cells in the blood target a particular organ and may enable the development of treatments to prevent the spread of cancers, known as metastasis.

In this study, Min Yu, assistant professor of stem cell and regenerative medicine at the Keck School of Medicine of USC, isolated breast cancer cells from the blood of breast cancer patients with metastatic tumors. Using a technique she developed previously, she expanded or grew the cells in the lab, creating a supply of material to work with.

Analyzing the tumor cells in animal models, Yus lab identified regulator genes and proteins within the cells that apparently directed the cancers spread to the brain. To test this concept, human tumor cells were injected into the bloodstream of animal models. As predicted, the cells migrated to the brain. Additional analysis of cells from one patients tumor predicted that the cells would later spread to the patients brain and they did.

Yu also discovered that a protein on the surface of brain-targeting tumor cells helps them to breach the blood-brain barrier and lodge in brain tissue, while another protein inside the cells shield them from the brains immune response, enabling them to grow there.

We can imagine someday using the information carried by circulating tumor cells to improve the detection, monitoring and treatment of the spreading cancers, Yu said. A future therapeutic goal is to develop drugs that get rid of circulating tumor cells or target those molecular signatures to prevent the spread of cancer.

Yu is a member of the Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, and her laboratory is located in the USC Norris Comprehensive Cancer Center.

In addition to Yu, other authors of the study are Remi Klotz, Amal Thomas, Teng Teng, Sung Min Han, Oihana Iriondo, Lin Li, Alan Wang, Negeen Izadian, Matthew MacKay, Byoung-San Moon, Kevin J. Liu, Sathish Kumar Ganesan, Grace Lee, Diane S. Kang, Michael F. Press, Wange Lu, Janice Lu, Bodour Salhia and Frank Attenello, all of the Keck School of Medicine; Sara Restrepo-Vassalli, James Hicks and Andrew D. Smith of USC Dornsife College of Letters, Arts and Sciences; and Charlotte S. Walmsley, Christopher Pinto, Dejan Juric and Aditya Bardia of Massachusetts General Hospital.

The study was supported by grants from the National Institutes of Health (DP2 CA206653) the Donald E. and Delia B. Baxter Foundation, the Stop Cancer Foundation, the Pew Charitable Trusts and the Alexander & Margaret Stewart Trust, the SC CTSI pilot grant (UL1TR001855 and UL1TR000130), a California Institute for Regenerative Medicine (CIRM) postdoctoral fellowship and a CIRM Bridges award (EDUC2-08381), and the National Cancer Institute (P30CA014089).

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Both Merus N.V. (NASDAQ:MRUS) and Sage Therapeutics Inc. (NASDAQ:SAGE) are Biotechnology companies, competing one another. We will compare their dividends, analyst recommendations, institutional ownership, profitability, risk, earnings and valuation.

Valuation and Earnings

Table 1 highlights Merus N.V. and Sage Therapeutics Inc.s gross revenue, earnings per share and valuation.

Profitability

Table 2 has Merus N.V. and Sage Therapeutics Inc.s return on equity, return on assets and net margins.

Liquidity

Merus N.V.s Current Ratio is 6.4 while its Quick Ratio is 6.4. On the competitive side is, Sage Therapeutics Inc. which has a 20.1 Current Ratio and a 20.1 Quick Ratio. Sage Therapeutics Inc. is better positioned to pay off short and long-term obligations compared to Merus N.V.

Analyst Ratings

The table shown features the ratings and recommendations for Merus N.V. and Sage Therapeutics Inc.

Merus N.V.s consensus price target is $20, while its potential upside is 22.17%. Competitively the consensus price target of Sage Therapeutics Inc. is $195, which is potential 39.59% upside. The information presented earlier suggests that Sage Therapeutics Inc. looks more robust than Merus N.V. as far as analyst view.

Institutional & Insider Ownership

Roughly 65.8% of Merus N.V. shares are owned by institutional investors while 98.75% of Sage Therapeutics Inc. are owned by institutional investors. Insiders owned 30.47% of Merus N.V. shares. Competitively, insiders own roughly 1.2% of Sage Therapeutics Inc.s shares.

Performance

Here are the Weekly, Monthly, Quarterly, Half Yearly, Yearly and YTD Performance of both pretenders.

For the past year Merus N.V. has weaker performance than Sage Therapeutics Inc.

Summary

On 6 of the 11 factors Merus N.V. beats Sage Therapeutics Inc.

Merus N.V., a clinical-stage immuno-oncology company, engages in developing bispecific antibody therapeutics. Its lead bispecific antibody candidate is MCLA-128, which is in Phase I/II clinical trials in Europe for the treatment of various solid tumors, including breast, gastric, and ovarian cancers. The company also develops MCLA-117, a bispecific antibody candidate that is expected to commence a Phase I/II clinical trial for the treatment of patients with acute myeloid leukemia, as well as for the treatment of myelodysplastic syndrome in pre-clinical studies, as well as developing MCLA-158, a bispecific antibody candidate, which is designed to bind to cancer stem cells for the potential treatment of colorectal cancer. Its pre-clinical bispecific antibody candidates include MCLA-134 and MCLA-145, as well as other early research projects. The company has a strategic collaboration with Incyte and ONO Pharmaceutical Co., Ltd. to develop bispecific antibody candidates based on Biclonics technology platform. Merus N.V. was founded in 2003 and is headquartered in Utrecht, the Netherlands.

Sage Therapeutics, Inc., a clinical-stage biopharmaceutical company, develops and commercializes novel medicines to treat central nervous system disorders. Its lead product candidate includes SAGE-547, a proprietary intravenous formulation of allopregnanolone that is in Phase III clinical development as an adjunctive therapy for the treatment of super-refractory status epilepticus (SRSE), as well as for the treatment of post-partum depression (PPD). The companys product pipeline includes SAGE-217, a novel neuroactive steroid, which is in Phase II clinical trials for the treatment of PPD, major depressive disorders, essential tremor, and Parkinsons diseases; and SAGE-689 a novel positive allosteric modulator of GABAA receptors that is in preclinical stage for the treatment of status epilepticus. In addition, its product pipeline that are in preclinical stage comprises SAGE-105, a novel neuroactive steroid for the treatment of orphon epilepsies; SAGE-324, a novel neuroactive steroid for the treatment of GABA hypofunction; and SAGE-718, a novel oxysterol-based positive allosteric modulator of NMDA receptors for the treatment of cerebrosterol deficit disorders, anti-NMDA receptor encephalitis, and NMDA hypofunction. The company was formerly known as Sterogen Biopharma, Inc. and changed its name to Sage Therapeutics, Inc. in September 2011. Sage Therapeutics, Inc. was founded in 2010 and is headquartered in Cambridge, Massachusetts.

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Comparison of Merus N.V. (MRUS) and Sage Therapeutics Inc. (NASDAQ:SAGE) - MS Wkly

As Biotechnology businesses, Cellectis S.A. (NASDAQ:CLLS) and Magenta Therapeutics Inc. (NASDAQ:MGTA), are affected by compare. This especially applies to their risk, analyst recommendations, institutional ownership, profitability, dividends, earnings and valuation.

Earnings & Valuation

Table 1 showcases the gross revenue, earnings per share and valuation of Cellectis S.A. and Magenta Therapeutics Inc.

Profitability

Table 2 hightlights the return on assets, net margins and return on equity of the two companies.

Liquidity

9.8 and 9.7 are the respective Current Ratio and a Quick Ratio of Cellectis S.A. Its rival Magenta Therapeutics Inc.s Current and Quick Ratios are 17.1 and 17.1 respectively. Magenta Therapeutics Inc. has a better chance of clearing its pay short and long-term debts than Cellectis S.A.

Insider and Institutional Ownership

Cellectis S.A. and Magenta Therapeutics Inc. has shares held by institutional investors as follows: 31.4% and 85.4%. Insiders Competitively, held 2.2% of Magenta Therapeutics Inc. shares.

Performance

In this table we provide the Weekly, Monthly, Quarterly, Half Yearly, Yearly and YTD Performance of both pretenders.

For the past year Cellectis S.A. had bearish trend while Magenta Therapeutics Inc. had bullish trend.

Summary

Cellectis S.A. beats Magenta Therapeutics Inc. on 7 of the 9 factors.

Cellectis S.A., a gene-editing company, develops and sells immuno-oncology products based on gene-edited T-cells that express chimeric antigen receptors to target and eradicate cancer in France. The company operates through two segments, Therapeutics and Plants. Its lead product candidate is UCART19, an allogeneic T-cell product candidate for the treatment of CD19 expressing hematologic malignancies, which develop in acute lymphoblastic leukemia (ALL) and CLL. The companys products also comprise UCART123 for acute myeloid leukemia indications and blastic plasmacytoid dendritic cell neoplasm; UCARTCS1 for multiple myeloma (MM) indications; UCART22 for ALL; and UCART38 for T-cell ALL and MM. In addition, it focuses on applying its gene-editing technologies to develop new generation plant products in the field of agricultural biotechnology. The company has strategic alliances with Pfizer Inc. to generate CAR T-cells in the field of oncology; Les Laboratoires Servier SAS to develop and commercialize product candidates; The University of Texas MD Anderson Cancer Center to research and develop novel cellular immunotherapies for patients suffering from various liquid tumors; and Cornell University to accelerate the development of a targeted immunotherapy for patients with acute myeloid leukemia. Cellectis S.A. was founded in 1999 and is headquartered in Paris, France.

Magenta Therapeutics, Inc., a clinical-stage biopharmaceutical company, engages in developing medicines to bring the curative power of bone marrow transplant to patients. It is developing C100, C200, and C300 targeted antibody-drug conjugates for transplant conditioning; MGTA-145, a stem cell mobilization product candidate to control stem cell mobilization; MGTA-456, an allogeneic stem cell therapy to control stem cell growth; E478, a small molecule aryl hydrocarbon receptor antagonist for the expansion of gene-modified stem cells; and G100, an ADC program to prevent acute graft and host diseases. The company was formerly known as HSCTCo Therapeutics, Inc. and changed its name to Magenta Therapeutics, Inc. in February 2016. Magenta Therapeutics, Inc. was incorporated in 2015 and is based in Cambridge, Massachusetts.

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Reviewing Cellectis S.A. (CLLS)'s and Magenta Therapeutics Inc. (NASDAQ:MGTA)'s results - MS Wkly

James F. Battey, Jr., MD, PhD; Laura K. Cole, PhD; and Charles A. Goldthwaite, Jr., PhD.

Stem cells are distinguished from other cells by two characteristics: (1) they can divide to produce copies of themselves (self-renewal) under appropriate conditions and (2) they are pluripotent, or able to differentiate into any of the three germ layers: the endoderm (which forms the lungs, gastrointestinal tract, and interior lining of the stomach), mesoderm (which forms the bones, muscles, blood, and urogenital tract), and ectoderm (which forms the epidermal tissues and nervous system). Pluripotent cells, which can differentiate into any mature cell type, are distinct from multipotent cells (such as hematopoietic, or blood-forming, cells) that can differ into a limited number of mature cell types. Because of their pluripotency and capacity for self-renewal, stem cells hold great potential to renew tissues that have been damaged by conditions such as type 1 diabetes, Parkinson's disease, heart attacks, and spinal cord injury. Although techniques to transplant multipotent or pluripotent cells are being developed for many specific applications, some procedures are sufficiently mature to be established options for care. For example, human hematopoietic cells from the umbilical cord and bone marrow are currently being used to treat patients with disorders that require replacement of cells made by the bone marrow, including Fanconi's anemia and chemotherapy-induced bone marrow failure after cancer treatment.

However, differentiation is influenced by numerous factors, and investigators are just beginning to understand the fundamental properties of human pluripotent cells. Researchers are gradually learning how to direct these cells to differentiate into specialized cell types and to use them for research, drug discovery, and transplantation therapy (see Figure 8.1). However, before stem cell derivatives are suitable for clinical application, scientists require a more complete understanding of the molecular mechanisms that drive pluripotent cells into differentiated cells. Scientists will need to pilot experimental transplantation therapies in animal model systems to assess the safety and long-term stable functioning of transplanted cells. In particular, they must be certain that any transplanted cells do not continue to self-renew in an unregulated fashion after transplantation, which may result in a teratoma, or stem cell tumor. In addition, scientists must ascertain that cells transplanted into a patient are not recognized as foreign by the patient's immune system and rejected.

Figure 8.1. The Scientific Challenge of Human Stem CellsThe state of the science currently lies in the development of fundamental knowledge of the properties of human pluripotent cells. The scientific capacity needs to be built, an understanding of the molecular mechanisms that drive cell specialization needs to be advanced, the nature and regulation of interaction between host and transplanted cells needs to be explored and understood, cell division needs to be understood and regulated, and the long-term stability of the function in transplanted cells needs to be established.

Stem cells derived from an early-stage human blastocyst (an embryo fertilized in vitro and grown approximately five days in culture) have the capacity to renew indefinitely, and can theoretically provide an unlimited supply of cells. It is also possible to derive stem cells from non-embryonic tissues, including amniotic fluid, placenta, umbilical cord, brain, gut, bone marrow, and liver. These stem cells are sometimes called "adult" stem cells, and they are typically rare in the tissue of origin. For example, blood-forming (hematopoietic) stem cell experts estimate that only 1 in 2000 to fewer than 1 in 10,000 cells found in the bone marrow is actually a stem cell.1 Because so-called "adult" stem cells include cells from the placenta and other early stages of development, they are more correctly termed "non-embryonic stem cells." Non-embryonic stem cells are more limited in their capacity to self renew in the laboratory, making it more difficult to generate a large number of stem cells for a specific experimental or therapeutic application. Under normal conditions, non-embryonic stem cells serve as a repair pool for the body, so they typically differentiate only into the cell types found in the organ of origin. Moreover, there is little compelling evidence for trans-differentiation, whereby a stem cell from one organ differentiates into a mature cell type of a different organ. New discoveries may overcome these limitations of stem cells derived from non-embryonic sources, and research directed toward this goal is currently underway in a number of laboratories.

Cultures of human pluripotent, self-renewing cells enable researchers to understand the molecular mechanisms that regulate differentiation (see Figure 8.2), including epigenetic changes (traits that may be inherited that do not arise from changes in the DNA sequence) in the chromatin structure, developmental changes in gene expression, exposure to growth factors, and interactions between adjacent cells. Understanding these basic mechanisms may enable future scientists to mobilize and differentiate endogenous populations of pluripotent cells to replace a cell type ravaged by injury or disease. Alternatively, scientists may some day be able to coax human pluripotent cells grown in the laboratory to become a specific type of specialized cell, which physicians could subsequently transplant into a patient to replace cells damaged by these same disease processes.

Scientists are gradually learning to direct the differentiation of pluripotent cell cultures into a specific type of cell, which can then be used as cellular models of human disease for drug discovery or toxicity studies. While it is not possible to predict the myriad ways that a basic understanding of stem cell differentiation may lead to new approaches for treating patients with cellular degenerative diseases, some avenues can be theorized. For example, in the case of Huntington's disease, a fatal neurodegenerative disorder, one could imagine that pluripotent cells derived from an embryo that carries Huntington's disease and differentiated into neurons in culture could be used to test drugs to delay or prevent degeneration.

Despite the incredible growth in knowledge that has occurred in stem cell research within the last couple of decades, investigators are just beginning to unravel the process of differentiation. Human pluripotent cell lines are an essential tool to understand this process and to facilitate the ultimate use of these cells in the clinic. To provide background on this fundamental topic, this article reviews the various potential sources and approaches that have been used to generate human pluripotent and multipotent cell lines, both of embryonic and non-embryonic origin.

Currently, at least six embryonic sources have been used to establish human pluripotent stem cell lines. All approaches involve isolation of viable cells during an early phase of development, followed by growth of these cells in appropriate culture medium. The various sources of these initial cell populations are discussed in brief below. It should be noted that the manipulation and use of embryonic tissues has raised a number of ethical issues.2,3 This article focuses on the scientific and technical issues associated with creating pluripotent cells, with the understanding that some of these techniques are currently subject to debates that extend beyond discussions of their scientific merits.

Figure 8.2. The Promise of Stem Cell ResearchStem cell research provides a useful tool for unraveling the molecular mechanisms that determine the differentiation fate of a pluripotent cell and for understanding the gene expression properties and epigenetic modifications essential to maintain the pluripotent state. In the future, this knowledge may be used to generate cells for transplantation therapies, whereby a specific cell population compromised by disease is replaced with new, functional cells. Differentiated derivatives of human pluripotent cells may also prove to be useful as models for understanding the biology of disease and developing new drugs, particularly when there is no animal model for the disease being studied. The greatest promise of stem cell research may lie in an area not yet imagined.

2008 Terese Winslow

Drawing upon twenty years of communal expertise with mouse ES cells,4 and on human inner cell mass culture conditions developed by Ariff Bongso and colleagues,5 James Thomson and colleagues at the University of Wisconsin generated the first hESC lines in 1998 using tissue from embryos fertilized in vitro.6 This method uses embryos generated for in vitro fertilization (IVF) that are no longer needed for reproductive purposes. During IVF, medical professionals usually produce more embryos than a couple attempting to start a family may need. Spare embryos are typically stored in a freezer to support possible future attempts for additional children if desired. It is estimated that there are approximately 400,000 such spare embryos worldwide.6 If these embryos are never used by the couple, they either remain in storage or are discarded as medical waste. Alternatively, these embryos can potentially be used to generate a hESC line.

To generate a hESC line, scientists begin with a donated blastocyst-stage embryo, at approximately five days after IVF (see Figure 8.3a). The blastocyst consists of approximately 150200 cells that form a hollow sphere of cells, the outer layer of which is called the trophectoderm. During normal development, the trophoblast becomes the placenta and umbilical cord. At one pole of this hollow sphere, 3050 cells form a cluster that is called the inner cell mass (ICM), which would give rise to the developing fetus. ICM cells are pluripotent, possessing the capacity to become any of the several hundred specialized cell types found in a developed human, with the exception of the placenta and umbilical cord.

Scientists remove the ICM from the donated blastocyst and place these cells into a specialized culture medium. In approximately one in five attempts, a hESC line begins to grow. Stem cells grown in such a manner can then be directed to differentiate into various lineages, including neural precursor cells,8 cardiomyocytes,9 and hematopoietic (blood forming) precursor cells.10

However, hESC lines are extremely difficult to grow in culture; the cells require highly specialized growth media that contain essential ingredients that are difficult to standardize. Yet the culture conditions are critical to maintain the cells' self-renewing and pluripotent properties. Culture requires the support of mouse or human cells, either directly as a "feeder" cell layer6,11,12 or indirectly as a source of conditioned medium in feeder-free culture systems.13 The feeder cells secrete important nutrients and otherwise support stem cell growth, but are treated so they cannot divide. Although the complete role of these feeder cells is not known, they promote stem cell growth, including detoxifying the culture medium and secreting proteins that participate in cell growth.14 hESC lines used to produce human cells for transplantation therapies may need to be propagated on a human feeder cell layer to reduce the risk of contamination by murine viruses or other proteins that may cause rejection. Thus, hESC lines often grow only under highly specific culture conditions, and the identification of ideal growth conditions presents a challenge regardless of the source of the hESCs.

Furthermore, human ES cell cultures must be expanded using an exacting protocol to avoid cell death and to control spontaneous differentiation. Since a limited number of laboratories in the United States are growing these cells, there is a shortage of people well-versed in the art and science of successful hESC culture. In the short term, challenges of working with these cells include developing robust culture conditions and protocols, understanding the molecular mechanisms that direct differentiation into specific cell types, and developing the infrastructure to advance this scientific opportunity. Once these challenges have been met, scientists will need to conduct transplantation studies in animal models (rodent and non-human primates) to demonstrate safety, effectiveness, and long-term benefit before stem cell therapies may enter clinical trials.

A second method for generating human pluripotent stem cell lines was published in 1998 by John Gearhart and coworkers at The Johns Hopkins Medical School.15

These researchers isolated specialized cells known as primordial germ cells (PGCs) from a 57-week-old embryo and placed these cells into culture (see Figure 8.3b). PGCs are destined to become either oocytes or sperm cells, depending on the sex of the developing embryo. The resulting cell lines are called embryonic germ cell lines, and they share many properties with ES cells. As with ES cells, however, PGCs present challenges with sustained growth in culture.16,17 Spontaneous differentiation, which hinders the isolation of pure clonal lines, is a particular issue. Therefore, the clinical application of these cells requires a more complete understanding of their derivation and maintenance in vitro.

Embryos that stop dividing after being fertilized in vitro are not preferentially selected for implantation in a woman undergoing fertility treatment. These embryos are typically either frozen for future use or discarded as medical waste. In 2006, scientists at the University of Newcastle, United Kingdom, generated hESC lines from IVF embryos that had stopped dividing.18 These scientists used similar methods as described under "Traditional hESC Line Generation" except that their source material was so-called "dead" IVF embryos (see Figure 8.3c). The human stem cells created using this technique behaved like pluripotent stem cells, including producing proteins critical for "stemness" and being able to produce cells from all three germ layers. It has been proposed that an IVF embryo can be considered dead when it ceases to divide.19 If one accepts this definition, such an embryo that "dies" from natural causes presumably cannot develop into a human being, thereby providing a source to derive human ES cells without destroying a living embryo.

Figure 8.3. Alternative Methods for Preparing Pluripotent Stem Cells

2008 Terese Winslow

Couples who have learned that they carry a genetic disorder sometimes use pre-implantation genetic diagnosis (PGD) and IVF to have a child that does not carry the disorder. PGD requires scientists to remove one cell from a very early IVF human embryo and test it for diseases known to be carried by the hopeful couple. Normally, embryos identified with genetic disorders are discarded as medical waste. However, Dr.Yuri Verlinsky and colleagues have capitalized on these embryos as a way to further our understanding of the diseases they carry (see Figure 8.3d) by deriving hESC lines from them.20 These stem cell lines can then be used to help scientists understand genetically-based disorders such as muscular dystrophy, Huntington's disease, thalessemia, Fanconi's anemia, Marfan syndrome, adrenoleukodystrophy, and neurofibromatosis.

In 2006, Dr. Robert Lanza and colleagues demonstrated that it is possible to remove a single cell from a pre-implantation mouse embryo and generate a mouse ES cell line.21 This work was based upon their experience with cleavage-stage mouse embryos. Later that same year, Dr. Lanza's laboratory reported that it had successfully established hESC lines (see Figure 8.3e) from single cells taken from pre-implantation human embryos.22 The human stem cells created using this technique behaved like pluripotent stem cells, including making proteins critical for "stemness" and producing cells from all three germ layers. Proponents of this technique suggest that since it requires only one embryonic cell, the remaining cells may yet be implanted in the womb and develop into a human being. Therefore, scientists could potentially derive human embryonic stem cells without having to destroy an embryo. However, ethical considerations make it uncertain whether scientists will ever test if the cells remaining after removal of a single cell can develop into a human being, at least in embryos that are not at risk for carrying a genetic disorder. Moreover, it is unclear whether the single cell used to generate a pluripotent stem cell line has the capacity to become a human being.

Parthenogenesis is the creation of an embryo without fertilizing the egg with a sperm, thus omitting the sperm's genetic contributions. To achieve this feat, scientists "trick" the egg into believing it is fertilized, so that it will begin to divide and form a blastocyst (see Figure 8.3f). In 2007, Dr. E.S. Revazova and colleagues reported that they successfully used parthenogenesis to derive hESCs.23 These stem cell lines, derived and grown using a human feeder cell layer, retained the genetic information of the egg donor and demonstrated characteristics of pluripotency. This technique may lead to the ability to generate tissue-matched cells for transplantation to treat women who are willing to provide their own egg cells.24 It also offers an alternate method for deriving tissue-matched hESCs that does not require destruction of a fertilized embryo.

Amniotic fluid surrounding the developing fetus contains cells shed by the fetus and is regularly collected from pregnant women during amniocentesis. In 2003, researchers identified a subset of cells in amniotic fluid that express Oct-4, a marker for pluripotent human stem cells that is expressed in ES cells and embryonic germ cells.25 Since then, investigators have shown that amniotic fluid stem cells can differentiate into cells of all three embryonic germ layers and that these cells do not form tumors in vivo.26,27

For example, Anthony Atala and colleagues at the Wake Forest University have recently generated non-embryonic stem cell lines from cells found in human and rat amniotic fluid.27 They named these cells amniotic fluid-derived stem cells (AFS). Experiments demonstrate that AFS can produce cells that originate from each of the three embryonic germ layers, and the self-renewing cells maintained the normal number of chromosomes after a prolonged period in culture. However, undifferentiated AFS did not produce all of the proteins expected of pluripotent cells, and they were not capable of forming a teratoma. The scientists developed in vitro conditions that enabled AFS to produce nerve cells, liver cells, and bone-forming cells. AFS-derived human nerve cells could make proteins typical of specialized nerve cells and were able to integrate into a mouse brain and survive for at least two months. Cultured AFS-derived human liver cells secreted urea and made proteins characteristic of normal human liver cells. Cultured AFS-derived human bone cells made proteins expected of human bone cells and formed bone in mice when seeded onto scaffolds and implanted under the mouse's skin. Although scientists do not yet know how many different cell types AFS can generate, AFS may one day allow researchers to establish a bank of cells for transplantation into humans.

An alternative to searching for an existing population of stem cells is to create a new one from a population of non-pluripotent cells. This strategy, which may or may not involve the creation of an embryo, is known as "reprogramming." This section will summarize reprogramming approaches, including several recent breakthroughs in the field..

In SCNT (see Figure 8.3g), human oocytes (eggs) are collected from a volunteer donor who has taken drugs that stimulate the production of more than one oocyte during the menstrual cycle. Scientists then remove the nucleus from the donated oocyte and replace it with the nucleus from a somatic cell, a differentiated adult cell from elsewhere in the body. The oocyte with the newly-transferred nucleus is then stimulated to develop. The oocyte may develop only if the transplanted nucleus is returned to the pluripotent state by factors present in the oocyte cytoplasm. This alteration in the state of the mature nucleus is called nuclear reprogramming. When development progresses to the blastocyst stage, the ICM is removed and placed into culture in an attempt to establish a pluripotent stem cell line. To date, the technique has been successfully demonstrated in two primates: macaque monkeys28 and humans.29

However, successful SCNT creates an embryo-like entity, thereby raising the ethical issues that confront the use of spare IVF embryos. However, pluripotent cell lines created by embryos generated by SCNT offer several advantages over ES cells. First, the nuclear genes of such a pluripotent cell line will be identical to the genes in the donor nucleus. If the nucleus comes from a cell that carries a mutation underlying a human genetic disease such as Huntington's disease, then all cells derived from the pluripotent cell line will carry this mutation. In this case, the SCNT procedure would enable the development of cellular models of human genetic disease that can inform our understanding of the biology of disease and facilitate development of drugs to slow or halt disease progression. Alternatively, if the cell providing the donor nucleus comes from a specific patient, all cells derived from the resulting pluripotent cell line will be genetically matched to the patient with respect to the nuclear genome. If these cells were used in transplantation therapy, the likelihood that the patient's immune system would recognize the transplanted cells as foreign and initiate tissue rejection would be reduced. However, because mitochondria also contain DNA, the donor oocyte will be the source of the mitochondrial genome, which is likely to carry mitochondrial gene differences from the patient which may still lead to tissue rejection.

A technique reported in 2007 by Dr. Kevin Eggan and colleagues at Harvard University may expand scientists' options when trying to "reprogram" an adult cell's DNA30. Previously, successful SCNT relied upon the use of an unfertilized egg. Now, the Harvard scientists have demonstrated that by using a drug to stop cell division in a fertilized mouse egg (zygote) during mitosis, they can successfully reprogram an adult mouse skin cell by taking advantage of the "reprogramming factors" that are active in the zygote at mitosis. They removed the chromosomes from the single-celled zygote's nucleus and replaced them with the adult donor cell's chromosomes (see Figure 8.3h). The active reprogramming factors present in the zygote turned genes on and off in the adult donor chromosomes, to make them behave like the chromosomes of a normally fertilized zygote. After the zygote was stimulated to divide, the cloned mouse embryo developed to the blastocyst stage, and the scientists were able to harvest embryonic stem cells from the resulting blastocyst. When the scientists applied their new method to abnormal mouse zygotes, they succeeded at reprogramming adult mouse skin cells and harvesting stem cells. If this technique can be repeated with abnormal human zygotes created in excess after IVF procedures, scientists could use them for research instead of discarding them as medical waste.

Altered nuclear transfer is a variation on standard SCNT that proposes to create patient-specific stem cells without destroying an embryo. In ANT, scientists turn off a gene needed for implantation in the uterus (Cdx2) in the patient cell nucleus before it is transferred into the donor egg (see Figure 8.3i). In 2006, Dr. Rudolph Jaenisch and colleagues at MIT demonstrated that ANT can be carried out in mice.31 Mouse ANT entities whose Cdx2 gene is switched off are unable to implant in the uterus and do not survive to birth. Although ANT has been used to create viable stem cell lines capable of producing almost all cell types, the authors point out that this technique must still be tested with monkey and human embryos. Moreover, the manipulation needed to control Cdx2 expression introduces another logistical hurdle that may complicate the use of ANT to derive embryonic stem cells. Proponents of ANT, such as William Hurlbut of the Stanford University Medical Center, suggest that the entity created by ANT is not a true embryo because it cannot implant in the uterus.32, 33 However, the technique is highly controversial, and its ethical implications remain a source of current debate.4,32

In 2005, Kevin Eggan and colleagues at Harvard University reported that they had fused cultured adult human skin cells with hESCs (see Figure 8.3j).36 The resulting "hybrid" cells featured many characteristics of hESCs, including a similar manner of growth and division and the manufacture of proteins typically produced by hESCs. Some factor(s) within the hESCs enabled them to "reprogram" the adult skin cells to behave as hESCs. However, these cells raised a significant technical barrier to clinical use. Because fused cells are tetraploid (they contain four copies of the cellular DNA rather than the normal two copies), scientists would need to develop a method to remove the extra DNA without eliminating their hESC-like properties. The fusion method serves as a useful model system for studying how stem cells "reprogram" adult cells to have properties of pluripotent cells. However, if the reprogramming technique could be carried out without the fusion strategy, a powerful avenue for creating patient-specific stem cells without using human eggs could be developed.

In 2007, two independent research groups published manuscripts that described successful genetic reprogramming of human adult somatic cells into pluripotent human stem cells.34,35 Although some technical limitations remain, this strategy suggests a promising new avenue for generating pluripotent cell lines that can inform drug development, models of disease, and ultimately, transplantation medicine. These experiments, which are discussed below, were breakthroughs because they used adult somatic cells to create pluripotent stem cells that featured hallmarks of ES cells.

In 2006, Shinya Yamanaka and colleagues at Kyoto University reported that they could use a retroviral expression vector to introduce four important stem cell factors into adult mouse cells and reprogram them to behave like ES cells (see Figure 8.3k).37 They called the reprogrammed cells "iPSCs," for induced pluripotent stem cells. However, iPSCs produced using the original technique failed to produce sperm and egg cells when injected into an early mouse blastocyst and did not make certain critical DNA changes. These researchers then modified the technique to select for iPSCs that can produce sperm and eggs,38 results that have since been reproduced by Rudolph Jaenisch and colleagues at the Massachusetts Institute of Technology (MIT).39

In addition, the MIT scientists determined that iPSCs DNA is modified in a manner similar to ES cells, and important stem cell genes are expressed at similar levels. They also demonstrated that iPSCs injected into an early mouse blastocyst can produce all cell types within the developing embryo, and such embryos can complete gestation and are born alive.

Once these research advances were made in mice, they suggested that similar techniques might be used to reprogram adult human cells. In 2007, Yamanaka and coworkers reported that introducing the same four genetic factors that reprogrammed the mouse cells into adult human dermal fibroblasts reprogrammed the cells into human iPSCs.35 These iPSCs were similar to human ES cells in numerous ways, including morphology, proliferative capacity, expression of cell surface antigens, and gene expression. Moreover, the cells could differentiate into cell types from the three embryonic germ layers both in vitro and in teratoma assays. Concurrent with the Yamanaka report, James Thomson and coworkers at the University of Wisconsin published a separate manuscript that detailed the creation of human iPSCs through somatic cell reprogramming using four genetic factors (two of which were in common with the Yamanaka report).34 The cells generated by the Thomson group met all defining criteria for ES cells, with the exception that they were not derived from embryos.

These breakthroughs have spurred interest in the field of iPSCs research. In early 2008, investigators at the Massachusetts General Hospital40 and the University of California, Los Angeles41 reported generating reprogrammed cells. As scientists explore the mechanisms that govern reprogramming, it is anticipated that more reports will be forthcoming in this emerging area. Although these reprogramming methods require the use of a virus, non-viral strategies may also be possible in the future. In any case, these approaches have created powerful new tools to enable the "dedifferentation" of cells that scientists had previously believed to be terminally differentiated.42,43

Although further study is warranted to determine if iPS and ES cells differ in clinically significant ways, these breakthrough reports suggest that reprogramming is a promising strategy for future clinical applications. Induced pluripotent cells offer the obvious advantage that they are not derived from embryonic tissues, thereby circumventing the ethical issues that surround use of these materials. Successful reprogramming of adult somatic cells could also lead to the development of stem cell lines from patients who suffer from genetically-based diseases, such as Huntington's Disease, spinal muscular atrophy, muscular dystrophy, and thalessemia. These lines would be invaluable research tools to understand the mechanisms of these diseases and to test potential drug treatments. Additionally, reprogrammed cells could potentially be used to repair damaged tissues; patient-specific cell lines could greatly reduce the concerns of immune rejection that are prevalent with many transplantation strategies.

However, several technical hurdles must be overcome before iPSCs can be used in humans. For example, in preliminary experiments with mice, the virus used to introduce the stem cell factors sometimes caused cancers.37 The viral vectors used in these experiments will have to be selected carefully and tested fully to verify that they do not integrate into the genome, thereby harboring the potential to introduce genetic mutations at their site of insertion. This represents a significant concern that must be addressed before the technique can lead to useful treatments for humans. However, this strategy identifies a method for creating pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

Stem cell research is a rapidly evolving field, and researchers continue to isolate new pluripotent cells and create additional cell lines. This section briefly reviews other sources of pluripotent cells and the implications that their discovery may have on future research.

Epiblast Cells. While rodent and human ES cells are pluripotent, they maintain their respective pluripotencies through different molecular signaling pathways. It is not known why these differences exist. Recently, several research groups have reported the generation of stable, pluripotent cell lines from mouse and rat epiblast, a tissue of the post-implantation embryo that ultimately generates the embryo proper.44,45 These cells are distinct from mouse ES cells in terms of the signals that control their differentiation. However, the cells share patterns of gene expression and signaling responses with human ES cells. The establishment of epiblast cell lines can therefore provide insight into the distinctions between pluripotent cells from different species and illuminate ways that pluripotent cells pursue distinct fates during early development.

Existing Adult Stem Cells. As has been discussed in other chapters, numerous types of precursor cells have been isolated in adult tissues.46 Although these cells tend to be relatively rare and are dispersed throughout the tissues, they hold great potential for clinical application and tissue engineering. For example, tissues created using stem cells harvested from an adult patient could theoretically be used clinically in that patient without engendering an immune response. Moreover, the use of adult stem cells avoids the ethical concerns associated with the use of ES cells. In addition, adult-derived stem cells do not spontaneously differentiate as do ES cells, thus eliminating the formation of teratomas often seen with implantation of ES cells. The potential of adult stem cells for regenerative medicine is great; it is likely that these various cells will find clinical application in the upcoming decades.

Although the recent advances in reprogramming of adult somatic cells has generated a wave of interest in the scientific community, these cell lines will not likely replace hESC lines as tools for research and discovery. Rather, both categories of cells will find unique uses in the study of stem cell biology and the development and evaluation of therapeutic strategies. Pluripotent cells offer a number of potential clinical applications, especially for diseases with a genetic basis. However, researchers are just beginning to unlock the many factors that govern the cells' growth and differentiation. As scientists make strides toward understanding how these cells can be manipulated, additional applications, approaches, and techniques will likely emerge. As such, pluripotent cells will play a pivotal role in future research into the biology of development and the treatment of disease.

Chapter7|Table of Contents|Chapter9

Excerpt from:Alternate Methods for Preparing Pluripotent Stem Cells ...

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Clinical study.

The clinical study titled 'Effects of Socioenvironmental Stress on the Human Hematopoietic System' was an open, monocenter, single-arm study that enrolled medical residents working on the intensive care unit at University Hospital, Freiburg, Germany. This study was registered with the German Registry for Clinical Studies (DRKS00004821) and was approved by the Ethics Committee of Albert-Ludwigs-University Freiburg, Germany (No. 52/13). All residents working on the ICU were considered eligible to participate in the study. Exclusion criteria were smoking, any acute or chronic illness, regular intake of medication or failure to consent. Twenty-nine volunteers (23 male, 6 female, mean age 33.7 0.8 years) were enrolled after signing the informed consent form. Residents gave two blood samples (baseline and stress). The off-duty sample (baseline) was collected after 10 0.9 consecutive days off duty. The on-duty sample (stress) was collected after 7 0.3 consecutive days of ICU duty. A subcohort of participants completed the Perceived Stress Scale 10-item inventory5 before starting to work on the ICU (baseline), as well as after several weeks on duty (stress). Short-term perception for stress frequency and intensity was measured with visual analog scales (scale 010)6, which each participant completed at the time of the blood sampling. The mean circadian time difference between the baseline and the stress sample was 20 15.9 min. Blood samples were analyzed in a blinded fashion at the routine clinical laboratory of the University Hospital, Freiburg, Germany.

We used C57BL/6, CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ), UBC-GFP (C57BL/6-Tg(UBC-GFP)30Scha/J), Apoe/ (B6.129P2-Apoetm1Unc/J), TH-Cre (B6.Cg-Tg(Th-Cre)1Tmd/J) and iDTR (C57BL/6-Gt(ROSA)26Sortm1(HBEGF)Awai/J) mice, all female and 1012 weeks of age (Jackson Laboratories, Bar Harbor, ME). Adrb3/ mice16 were donated by P. Frenette (Albert Einstein College of Medicine, New York, NY, USA) and B. Lowell (Beth Israel Deaconess Medical Center, Boston, MA, USA). Nestin-GFP mice41 were a gift from G. Enikolopov (Cold Spring Harbor Laboratory, NY). All procedures were approved by the Subcommittee on Animal Research Care at Massachusetts General Hospital. For each experiment, age-matched female littermates were randomly allocated to study groups. Animal studies were performed without blinding of the investigator.

Mice were exposed to socioenvironmental stressors7,8,9 for one or three weeks in C57BL/6 mice or six weeks in Apoe/ mice. Stress procedures were performed between 7 a.m. and 6 p.m. The following stressors were applied. For cage tilt, the cage was tilted at a 45 angle and kept in this position for six hours. For isolation, mice were individually housed in an area one-quarter of the original cage size (12 cm 8 cm) for four hours, followed by crowding, during which 10 animals were housed in one cage for two hours. Mice were monitored during the crowding procedure, and 'fighters' were separated. For damp bedding, water was added to the cage to moisten the bedding without generating large pools. Mice were kept for six hours with damp bedding. For rapid light-dark changes, using an automatic timer, the light was switched with an interval of seven minutes for two hours. For overnight illumination, mice were housed in a separate room with illumination from 7 p.m. to 7 a.m. All stressors were randomly shuffled in consecutive weeks. Efficacy of the chronic stress procedures was confirmed by measurement of blood corticosterone levels (Supplementary Fig. 12c).

Mice were irradiated using a split dose of 2 600 cGy with an interval of 3 h between doses. Animals were irradiated 12 h before bone marrow reconstitution.

For competitive bone marrow repopulation assays42, we co-transferred 2 106 whole bone marrow cells from CD45.1 mice after three weeks of stress or from nonstressed controls together with equal cell numbers of CD45.2 competitor cells from nonstressed wild-type mice into lethally irradiated UBC-GFP CD45.2 mice. Engraftment was assessed by comparing blood leukocyte chimerism for CD45.1 cells between groups after 2, 3 and 4 months. For limiting dilution experiments42, donor doses of 1.5 104, 6 104, 12.5 104 or 5 105 whole bone marrow cells from CD45.1 mice after three weeks of stress or from nonstressed controls were co-transferred with 5 105 CD45.2 competitor cells into lethally irradiated CD45.2 recipients. Engraftment was assessed after four months as at least >0.1% multilineage blood chimerism for B lymphocytes, T lymphocytes and myeloid lineage cells derived from donor bone marrow. Poisson's statistic was calculated using L-calc software (Stemcell Technologies) and ELDA software43. Bone marrow of two mice was pooled for each cell population.

To inhibit 3-adrenergic signaling, a specific antagonist for the 3-adrenergic receptor (SR 59230A, Sigma-Aldrich) was injected at 5 mg/kg body weight i.p. twice per day44. For inhibition of 2-adrenergic signaling, ICI118,551 hydrochloride (Sigma-Aldrich) was injected daily at a dose of 1 mg/kg body weight i.p. (ref. 18) for three weeks. The control groups received saline injections.

TH-Cre mice were cross-bred with iDTR mice. 1012 week old female TH-iDTR mice were intraperitoneally injected with 0.1 g/kg body weight diphtheria toxin (DT) on day 0 and day 3 after initiation of stress procedures18. Age-matched littermates (TH-Cre, iDTR or WT) that were also stressed and injected with DT served as controls.

Nonstressed mice and mice that had been stressed for three weeks were injected intravenously with 150 mg/kg body weight 5-FU (Sigma)45 on day 0. Mice were then followed over the course of 21 days, and the absolute number of blood leukocytes was measured after 7, 14 and 21 days. Stress exposure continued for the remaining 3 weeks after 5-FU exposure.

Flushed bone marrow was passed through a 40-m cell strainer and collected in PBS containing 0.5% BSA and 1% FBS (FACS buffer). Aortas were excised, minced and digested in collagenase I (450 U/ml), collagenase XI (125 U/ml), DNase I (60 U/ml) and hyaluronidase (60 U/ml) (all Sigma-Aldrich) at 37 C at 750 r.p.m. for 1 h. For sorting niche cells, bones were harvested from nestin-GFP mice. Bone marrow endothelial cells (ECs) and mesenchymal stem cells (MSCs) were obtained by flushing out bone marrow, which was then digested in 10 mg/ml collagenase type IV (Worthington) and 20 U/ml DNase I (Sigma)46. For obtaining bone osteoblastic lineage cells, we crushed bones, washed off residual bone marrow cells three times and then digested and incubated the bone fragments47,48.

For myeloid cells, cells were first stained with mouse hematopoietic lineage markers (1:600 dilution for all antibodies) including phycoerythrin (PE) anti-mouse antibodies directed against B220 (BD Bioscience, clone RA3-6B2), CD90 (BD Bioscience, clone 53-2.1), CD49b (BD Bioscience, clone DX5), NK1.1 (BD Bioscience, clone PK136) and Ter-119 (BD Bioscience, clone TER-119). This was followed by a second staining for CD45.2 (BD Bioscience, clone 104, 1:300), CD11b (BD Bioscience, clone M1/70, 1:600), CD115 (eBioscience, clone M1/70, 1:600), Ly6G (BD Bioscience, clone 1A8, 1:600), CD11c (eBioscience, clone HL3, 1:600), F4/80 (Biolegend, clone BM8, 1:600) and Ly6C (BD Bioscience, clone AL-21, 1:600). Neutrophils were identified as (CD90/B220/CD49b/NK1.1/Ter119)low(CD45.2/CD11b)highCD115lowLy6Ghigh. Monocytes were identified as (CD90/B220/CD49b/NK1.1/Ter119)lowCD11bhigh(F4/80/CD11c)lowLy-6Chigh/low or (CD45.2/CD11b)highLy6GlowCD115highLy-6Chigh/low. Macrophages were identified as (CD90/B220/CD49b/NK1.1/Ter119)lowCD11bhighLy6Clow/intLy6GlowF4/80high. For hematopoietic progenitor staining, we first incubated cells with biotin-conjugated anti-mouse antibodies (1:600 dilution for all antibodies) directed against B220 (eBioscience, clone RA3-6B2), CD11b (eBioscience, clone M1/70), CD11c (eBioscience, clone N418), NK1.1 (eBioscience, clone PK136), TER-119 (eBioscience, clone TER-119), Gr-1 (eBioscience, clone RB6-8C5), CD8a (eBioscience, clone 53-6.7), CD4 (eBioscience, clone GK1.5) and IL7R (eBioscience, clone A7R34) followed by pacific orangeconjugated streptavidin anti-biotin antibody. Then cells were stained with antibodies directed against c-Kit (BD Bioscience, clone 2B8, 1:600), Sca-1 (eBioscience, clone D7, 1:600), SLAM markers10 CD48 (eBioscience, clone HM48-1, 1:300) and CD150 (Biolegend, clone TC15-12F12.2, 1:300), CD34 (BD Bioscience, clone RAM34, 1:100), CD16/32 (BD Bioscience, clone 2.4G2, 1:600) and CD115 (eBioscience, clone AFS98, 1:600). LSKs were identified as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-KithighSca-1high. HSCs were identified as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-KithighSca-1highCD48lowCD150high. Granulocyte macrophage progenitors were defined as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-KithighSca-1low(CD34/CD16/32)highCD115int/low. Macrophage dendritic cell progenitors were defined as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4 IL7R)lowc-Kitint/highSca-1low(CD34/CD16/32)highCD115high. Common lymphoid progenitors were identified as (B220 CD11b CD11c NK1.1 Ter-119 Ly6G CD8a CD4)lowc-KitintSca-1intIL7Rhigh. For staining endothelial cells, we used ICAM-1 (Biolegend, clone Yn1/1.7.4, 1:300), ICAM-2 (Biolegend, clone 3C4, 1:300), VCAM-1 (Biolegend, clone 429, 1:300), E-selectin (CD62E) (BD Bioscience, clone 10E9.6, 1:100), P-selectin (CD62P) (BD Bioscience, clone RB40.34, 1:100), CD31 (Biolegend, clone 390, 1:600), CD107a (LAMP-1) (Biolegend, clone 1D4B, 1:600) and CD45.2 (Biolegend, clone 104, 1:300). Streptavidinpacific orange was used to label biotinylated antibodies. Endothelial cells were identified as CD45.2low, CD31high and CD107aintermed/high. For analysis of human monocyte subsets, cells were stained for HLA-DR (Biolegend, clone L243, 1:600), CD16 (Biolegend, clone 3G8, 1:600) and CD14 (Biolegend, clone HCD14, 1:600) after red blood cell lysis (RBC Lysis buffer, Biolegend). Monocytes were identified using forward and side scatter as well as HLA-DR. Within this population, frequencies of monocyte subsets CD14high, CD16high and CD14high/CD16high were quantified.

For BrdU pulse experiments, we used APC/FITC BrdU flow kits (BD Bioscience). One mg BrdU was injected i.p. 24 h before organ harvest. BrdU staining was performed according to the manufacturer's protocol. For BrdU application over 7 days, osmotic micropumps (Alzet) filled with 18mg BrdU were implanted. For the BrdU label-retaining pulse chase assay, BrdU was added to drinking water (1 mg/ml) for 17 days11.

After surface staining, intracellular staining was performed according to eBioscience's protocol: cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and then stained for the nuclear antigen Ki-67 (eBioscience, clone SolA15). Cell cycle status was determined using 4,6-diamidino-2-phenylindole (DAPI, FxCycle Violet Stain, Life Technologies).

To isolate HSPCs, we used MACS depletion columns (Miltenyi) after incubation with a cocktail of biotin-labeled antibodies (as described in the flow cytometry section) followed by incubation with streptavidin-coated microbeads (Miltenyi). Next, cells were stained with c-Kit and Sca-1, and LSKs were FACS-sorted using a FACSAria II cell sorter (BD Biosystems). To purify niche cells from hematopoietic cells, we used MACS depletion columns after incubation with a cocktail of biotin-labeled antibodies as above followed by incubation with streptavidin-coated microbeads. Cells were then stained with CD45.2, Sca-1, CD31 and CD51 (Biolegend, clone RMV-7, 1:100). Endothelial cells were identified as LinlowCD45lowSca-1highCD31high. Bone marrow MSCs were identified as LinlowCD45lowCD31lowSca-1high/intermediate and GFP+. Osteoblasts were LinlowCD45lowSca-1lowCD31lowCD51high. For adoptive transfer of GFP+ neutrophils and Ly6Chigh monocytes, bone marrow cells were collected from UBC-GFP mice for purification of neutrophils and monocytes using MACS depletion columns after incubation with a cocktail of PElabeled antibodies including B220, CD90, CD49b, NK1.1 and Ter-119 followed by an incubation with PE-coated microbeads. Aortic endothelial cells were identified as CD45.2lowCD31highCD107aint/high and FACS-sorted using a FACSAria II cell sorter.

We injected 2 106 neutrophils together with 2 106 Ly6Chigh monocytes intravenously into nonstressed and stressed Apoe/ mice (the mice were stressed for 6 weeks, and the cells were injected 2 days before the end of the 6 weeks). Aortas were harvested 48 h later. The number of CD11bhighGFP+ cells within the aorta was quantified using flow cytometry.

Aortic roots were harvested and embedded to produce 6-m sections that were stained using an anti-CD11b (BD Biosciences, clone M1/70, 1:15 dilution) or anti-Ly6G (Biolegend, clone 1A8, 1:25 dilution) antibody followed with a biotinylated secondary antibody. For color development, we used the VECTA STAIN ABC kit (Vector Laboratories, Inc.) and AEC substrate (DakoCytomation). Necrotic core and fibrous cap thickness were assessed using Masson trichrome (Sigma) staining. Necrotic core was evaluated by measuring the total acellular area within each plaque. For fibrous cap thickness, three to five measurements representing the thinnest part of the fibrous cap were averaged for each plaque as previously described49. For tyrosine hydroxylase staining, femurs were harvested and fixed in 4% paraformaldehyde for 3 h and then decalcified in 0.375 M EDTA in PBS for 10 days before paraffin embedding. Sections were cut and stained with antityrosine hydroxylase antibody (Millipore, AB152, dilution 1:100) after deparaffinization and rehydration. Sections were scanned with NanoZoomer 2.0-RS (Hamamatsu) at 40 magnification and analyzed using IPLab (Scanalytics).

For intravital microscopy of hematopoietic progenitors in the bone marrow of the calvarium, LSKs were isolated from either wild-type C57BL/6 or C57BL/6-Tg(UBC-GFP)30Scha/J mice and labeled with the lipophilic membrane dye DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine perchlorate, Invitrogen). 25,000 labeled LSKs were transferred i.v. into nonirradiated C57BL/6 recipient mice. For blood pool contrast, TRITCdextran (Sigma) was injected immediately before imaging. OsteoSense 750 (PerkinElmer) was injected i.v. 24 h before in vivo imaging to outline bone structures in the calvarium50. In vivo imaging was performed on days 1 and 7 after the adoptive cell transfer using an IV100 confocal microscope (Olympus)15. Three channels were recorded (DiD excitation/emission 644/665 nm, OsteoSense 750 excitation/emission 750/780 nm, TRITCDextran excitation/emission 557/576 nm) to generate z stacks of each location at 2-m steps. Image postprocessing was performed using Image J software. Mean DiD fluorescence intensity was measured for each labeled cell and then normalized to the background by calculating the target to background ratio.

Colony-forming unit (CFU) assays were performed using a semisolid cell culture medium (Methocult M3434, Stem Cell Technology) following the manufacturer's protocol. Bones were flushed with Iscove's Modified Dulbecco's Medium (Lonza) supplemented with 2% FCS. 2 104 bone marrow cells were plated on a 35-mm plate in duplicates and incubated for 7 days. Colonies were counted using a low magnification inverted microscope.

Blood pressure and heart rate were measured using a noninvasive tail-cuff system (Kent Scientific Corporation) according to the manufacturer's instructions. For each value, the mean of three consecutive measurements was used.

Messenger RNA (mRNA) was extracted from aortic arches or bone marrow using the RNeasy Mini Kit (Qiagen) or from FACS-sorted cells using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) according to the manufacturers' protocol. One microgram of mRNA was transcribed to complementary DNA (cDNA) with the high capacity RNA to cDNA kit (Applied Biosystems). We used Taqman primers (Applied Biosystems). Results were expressed by Ct values normalized to the housekeeping gene Gapdh.

After six weeks of stress, FMT-CT imaging was performed and compared to nonstressed, age-matched Apoe/ controls. Pan-cathepsin protease sensor (Prosense-680, PerkinElmer, 5 nmol) was injected intravenously 24 h before the imaging as previously described51.

Blood corticosterone levels were measured by ELISA (Abcam). Serum was collected between 10 a.m. and 12 p.m. For measurements of noradrenaline in the bone marrow, a 2CAT (AN) Research ELISA (Labor Diagnostika Nord) was used. One femur was snap-frozen and immediately homogenized in a catecholamine stabilizing solution containing sodium metabisulfite (4 mM), EDTA (1 mM) and hydrochloric acid (0.01 N). Prior to the ELISA, the pH of the sample was adjusted to 7.5 using sodium hydroxide (1 N). ELISAs for CXCL12 (R&D), IFN- (PBL Biomedical Laboratories) and IFN- (R&D) in the bone marrow were performed using one femur and one tibia per mouse14. ELISAs were performed according to the manufacturers' instructions.

Statistical analyses were performed using GraphPad Prism software (GraphPad Software, Inc.). Results are depicted as mean standard error of mean if not stated otherwise. For a two-group comparison, a Student's t-test was applied if the pretest for normality (D'Agostino-Pearson normality test) was not rejected at the 0.05 significance level; otherwise, a Mann-Whitney U test for nonparametric data was used. For a comparison of more than two groups, an ANOVA test, followed by a Bonferroni test for multiple comparison, was applied. For analysis of clinical data, a Wilcoxon test for matched pairs was used. P values of <0.05 indicate statistical significance. No statistical method was used to predetermine sample size.

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Chronic variable stress activates hematopoietic stem cells ...

Stem cells have the ability to repair, replace and or restore biological structures and functions that may be damaged due to aging or disease. Stem cell therapy, also referred to as regenerative medicine, is being studied both nationally and internationally in a broad range of diseases. Stem cell therapy is now being used to treat diseases in humans with a large number of studies being published each year documenting success. However, unfortunately for residents of Boston and Massachusetts as a whole, legal stem cell therapy is not currently available. However, in Costa Rica, stem cell treatment is legal, and the Stem Cells Transplant Institute in Costa Rica believes in the potential benefits of stem cell therapy and provides government approved treatments for: Parkinsons disease, Alzheimers disease, cardiovascular disease, multiple sclerosis, lupus, rheumatoid arthritis, osteoarthritis, knee injury, chronic obstructive pulmonary disease, myocardial infarction, and critical limb ischemia.

In the United States, medical companies and research institutions in Boston are collaborating locally and nationally to move stem cell therapy and regenerative medicine into clinical practice. The Harvard Stem Cell Institute, Massachusetts General Hospital and Boston Childrens Hospital are evaluating the efficacy of stem cell therapy in many areas including aging and degenerative disease but due to government regulations, progress in the United States is slow. Based on published data, clinics outside of the United States are treating patients with inflammatory and degenerative disease using stem cell therapy. Costa Rica is a leader in stem cell treatment and research.

Dr. Leslie Mesen, founder and CEO of the Stem Cells Transplant Institute, studied medicine at Universidad Iberoamericana (UNIBE) in San Jos, Costa Rica. Dr. Mesen is board certified in Anti-Aging and Regenerative Medicine byThe American Academy of Anti-Aging Medicineand receives continuing education courses in both the United States and Latin America. One of the top destinations for medical tourism, Costa Rica offers high quality care at affordable prices. Dont let aging or disease keep you from attending a New England Patriots game, enjoying a Boston Red Sox game at Fenway Park or attending the latest performance at the Boston Opera House. Let the experts at the Stem Cells Transplant Institute help you stay active and live your best life.

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Boston, MA, Stem Cell Transplant, Weston, Nantucket ...

Cloning/Embryonic Stem Cells

The term cloning is used by scientists to describe many different processes that involve making duplicates of biological material. In most cases, isolated genes or cells are duplicated for scientific study, and no new animal results. The experiment that led to the cloning of Dolly the sheep in 1997 was different: It used a cloning technique called somatic cell nuclear transfer and resulted in an animal that was a genetic twin -- although delayed in time -- of an adult sheep. This technique can also be used to produce an embryo from which cells called embryonic stem (ES) cells could be extracted to use in research into potential therapies for a wide variety of diseases.

Thus, in the past five years, much of the scientific and ethical debate about somatic cell nuclear transfer has focused on its two potential applications: 1) for reproductive purposes, i.e., to produce a child, or 2) for producing a source of ES cells for research.

The technique of transferring a nucleus from a somatic cell into an egg that produced Dolly was an extension of experiments that had been ongoing for over 40 years. In the simplest terms, the technique used to produce Dolly the sheep - somatic cell nuclear transplantation cloning - involves removing the nucleus of an egg and replacing it with the diploid nucleus of a somatic cell. Unlike sexual reproduction, during which a new organism is formed when the genetic material of the egg and sperm fuse, in nuclear transplantation cloning there is a single genetic "parent." This technique also differs from previous cloning techniques because it does not involve an existing embryo. Dolly is different because she is not genetically unique; when born she was genetically identical to an existing six-year-old ewe. Although the birth of Dolly was lauded as a success, in fact, the procedure has not been perfected and it is not yet clear whether Dolly will remain healthy or whether she is already experiencing subtle problems that might lead to serious diseases. Thus, the prospect of applying this technique in humans is troubling for scientific and safety reasons in addition to a variety of ethical reasons related to our ideas about the natural ordering of family and successive generations.

Several important concerns remain about the science and safety of nuclear transfer cloning using adult cells as the source of nuclei. To date, five mammalian species -- sheep, cattle, pigs, goats, and mice -- have been used extensively in reproductive cloning studies. Data from these experiments illustrate the problems involved. Typically, very few cloning attempts are successful. Many cloned animals die in utero, even at late stages or soon after birth, and those that survive frequently exhibit severe birth defects. In addition, female animals carrying cloned fetuses may face serious risks, including death from cloning-related complications.

An additional concern focuses on whether cellular aging will affect the ability of somatic cell nuclei to program normal development. As somatic cells divide they progressively age, and there is normally a defined number of cell divisions that can occur before senescence. Thus, the health effects for the resulting liveborn, having been created with an "aged" nucleus, are unknown. Recently it was reported that Dolly has arthritis, although it is not yet clear whether the five-and-a-half-year-old sheep is suffering from the condition as a result of the cloning process. And, scientists in Tokyo have shown that cloned mice die significantly earlier than those that are naturally conceived, raising an additional concern that the mutations that accumulate in somatic cells might affect nuclear transfer efficiency and lead to cancer and other diseases in offspring. Researchers working with clones of a Holstein cow say genetic programming errors may explain why so many cloned animals die, either as fetuses or newborns.

The announcement of Dolly sparked widespread speculation about a human child being created using somatic cell nuclear transfer. Much of the perceived fear that greeted this announcement centered on the misperception that a child or many children could be produced who would be identical to an already existing person. This fear is based on the idea of "genetic determinism" -- that genes alone determine all aspects of an individual -- and reflects the belief that a person's genes bear a simple relationship to the physical and psychological traits that compose that individual. Although genes play an essential role in the formation of physical and behavioral characteristics, each individual is, in fact, the result of a complex interaction between his or her genes and the environment within which he or she develops. Nonetheless, many of the concerns about cloning have focused on issues related to "playing God," interfering with the natural order of life, and somehow robbing a future individual of the right to a unique identity.

Several groups have concluded that reproductive cloning of human beings creates ethical and scientific risks that society should not tolerate. In 1997, the National Bioethics Advisory Commission recommended that it was morally unacceptable to attempt to create a child using somatic cell nuclear transfer cloning and suggested that a moratorium be imposed until safety of this technique could be assessed. The commission also cautioned against preempting the use of cloning technology for purposes unrelated to producing a liveborn child.

Similarly, in 2001 the National Academy of Sciences issued a report stating that the United States should ban human reproductive cloning aimed at creating a child because experience with reproductive cloning in animals suggests that the process would be dangerous for the woman, the fetus, and the newborn, and would likely fail. The report recommended that the proposed ban on human cloning should be reviewed within five years, but that it should be reconsidered "only if a new scientific review indicates that the procedures are likely to be safe and effective, and if a broad national dialogue on societal, religious and ethical issues suggests that reconsideration is warranted." The panel concluded that the scientific and medical considerations that justify a ban on human reproductive cloning at this time do not apply to nuclear transplantation to produce stem cells. Several other scientific and medical groups also have stated their opposition to the use of cloning for the purpose of producing a child.

The cloning debate was reopened with a new twist late in 1998, when two scientific reports were published regarding the successful isolation of human stem cells. Stem cells are unique and essential cells found in animals that are capable of continually reproducing themselves and renewing tissue throughout an individual organism's life. ES cells are the most versatile of all stem cells because they are less differentiated, or committed, to a particular function than adult stem cells. These cells have offered hope of new cures to debilitating and even fatal illness. Recent studies in mice and other animals have shown that ES cells can reduce symptoms of Parkinson's disease in mouse models, and work in other animal models and disease areas seems promising.

In the 1998 reports, ES cells were derived from in vitro embryos six to seven days old destined to be discarded by couples undergoing infertility treatments, and embryonic germ (EG) cells were obtained from cadaveric fetal tissue following elective abortion. A third report, appearing in the New York Times, claimed that a Massachusetts biotechnology company had fused a human cell with an enucleated cow egg, creating a hybrid clone that failed to progress beyond an early stage of development. This announcement served as a reminder that ES cells also could be derived from embryos created through somatic cell nuclear transfer, or cloning. In fact, several scientists believed that deriving ES cells in this manner is the most promising approach to developing treatments because the condition of in vitro fertilization (IVF) embryos stored over time is questionable and this type of cloning could overcome graft-host responses if resulting therapies were developed from the recipient's own DNA.

For those who believe that the embryo has the moral status of a person from the moment of conception, research or any other activity that would destroy it is wrong. For those who believe the human embryo deserves some measure of respect, but disagree that the respect due should equal that given to a fully formed human, it could be considered immoral not to use embryos that would otherwise be destroyed to develop potential cures for disease affecting millions of people. An additional concern related to public policy is whether federal funds should be used for research that some Americans find unethical.

Since 1996, Congress has prohibited researchers from using federal funds for human embryo research. In 1999, DHHS announced that it intended to fund research on human ES cells derived from embryos remaining after infertility treatments. This decision was based on an interpretation "that human embryonic stem cells are not a human embryo within the statutory definition" because "the cells do not have the capacity to develop into a human being even if transferred to the uterus, thus their destruction in the course of research would not constitute the destruction of an embryo." DHHS did not intend to fund research using stem cells derived from embryos created through cloning, although such efforts would be legal in the private sector.

In July 2001, the House of Representatives voted 265 to 162 to make any human cloning a criminal offense, including cloning to create an embryo for derivation of stem cells rather than to produce a child. In August 2002, President Bush, contending with a DHHS decision made during the Clinton administration, stated in a prime-time television address that federal support would be provided for research using a limited number of stem cell colonies already in existence (derived from leftover IVF embryos). Current bills before Congress would ban all forms of cloning outright, prohibit cloning for reproductive purposes, and impose a moratorium on cloning to derive stem cells for research, or prohibit cloning for reproductive purposes while allowing cloning for therapeutic purposes to go forward. As of late June, the Senate has taken no action. President Bush's Bioethics Council is expected to recommend the prohibition of reproductive cloning and a moratorium on therapeutic cloning later this summer.

Prepared by Kathi E. Hanna, M.S., Ph.D., Science and Health Policy Consultant

Last Reviewed: April 2006

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Cloning/Embryonic Stem Cells - National Human Genome ...

James F. Battey, Jr., MD, PhD; Laura K. Cole, PhD; and Charles A. Goldthwaite, Jr., PhD.

Stem cells are distinguished from other cells by two characteristics: (1) they can divide to produce copies of themselves (self-renewal) under appropriate conditions and (2) they are pluripotent, or able to differentiate into any of the three germ layers: the endoderm (which forms the lungs, gastrointestinal tract, and interior lining of the stomach), mesoderm (which forms the bones, muscles, blood, and urogenital tract), and ectoderm (which forms the epidermal tissues and nervous system). Pluripotent cells, which can differentiate into any mature cell type, are distinct from multipotent cells (such as hematopoietic, or blood-forming, cells) that can differ into a limited number of mature cell types. Because of their pluripotency and capacity for self-renewal, stem cells hold great potential to renew tissues that have been damaged by conditions such as type 1 diabetes, Parkinson's disease, heart attacks, and spinal cord injury. Although techniques to transplant multipotent or pluripotent cells are being developed for many specific applications, some procedures are sufficiently mature to be established options for care. For example, human hematopoietic cells from the umbilical cord and bone marrow are currently being used to treat patients with disorders that require replacement of cells made by the bone marrow, including Fanconi's anemia and chemotherapy-induced bone marrow failure after cancer treatment.

However, differentiation is influenced by numerous factors, and investigators are just beginning to understand the fundamental properties of human pluripotent cells. Researchers are gradually learning how to direct these cells to differentiate into specialized cell types and to use them for research, drug discovery, and transplantation therapy (see Figure 8.1). However, before stem cell derivatives are suitable for clinical application, scientists require a more complete understanding of the molecular mechanisms that drive pluripotent cells into differentiated cells. Scientists will need to pilot experimental transplantation therapies in animal model systems to assess the safety and long-term stable functioning of transplanted cells. In particular, they must be certain that any transplanted cells do not continue to self-renew in an unregulated fashion after transplantation, which may result in a teratoma, or stem cell tumor. In addition, scientists must ascertain that cells transplanted into a patient are not recognized as foreign by the patient's immune system and rejected.

Figure 8.1. The Scientific Challenge of Human Stem CellsThe state of the science currently lies in the development of fundamental knowledge of the properties of human pluripotent cells. The scientific capacity needs to be built, an understanding of the molecular mechanisms that drive cell specialization needs to be advanced, the nature and regulation of interaction between host and transplanted cells needs to be explored and understood, cell division needs to be understood and regulated, and the long-term stability of the function in transplanted cells needs to be established.

Stem cells derived from an early-stage human blastocyst (an embryo fertilized in vitro and grown approximately five days in culture) have the capacity to renew indefinitely, and can theoretically provide an unlimited supply of cells. It is also possible to derive stem cells from non-embryonic tissues, including amniotic fluid, placenta, umbilical cord, brain, gut, bone marrow, and liver. These stem cells are sometimes called "adult" stem cells, and they are typically rare in the tissue of origin. For example, blood-forming (hematopoietic) stem cell experts estimate that only 1 in 2000 to fewer than 1 in 10,000 cells found in the bone marrow is actually a stem cell.1 Because so-called "adult" stem cells include cells from the placenta and other early stages of development, they are more correctly termed "non-embryonic stem cells." Non-embryonic stem cells are more limited in their capacity to self renew in the laboratory, making it more difficult to generate a large number of stem cells for a specific experimental or therapeutic application. Under normal conditions, non-embryonic stem cells serve as a repair pool for the body, so they typically differentiate only into the cell types found in the organ of origin. Moreover, there is little compelling evidence for trans-differentiation, whereby a stem cell from one organ differentiates into a mature cell type of a different organ. New discoveries may overcome these limitations of stem cells derived from non-embryonic sources, and research directed toward this goal is currently underway in a number of laboratories.

Cultures of human pluripotent, self-renewing cells enable researchers to understand the molecular mechanisms that regulate differentiation (see Figure 8.2), including epigenetic changes (traits that may be inherited that do not arise from changes in the DNA sequence) in the chromatin structure, developmental changes in gene expression, exposure to growth factors, and interactions between adjacent cells. Understanding these basic mechanisms may enable future scientists to mobilize and differentiate endogenous populations of pluripotent cells to replace a cell type ravaged by injury or disease. Alternatively, scientists may some day be able to coax human pluripotent cells grown in the laboratory to become a specific type of specialized cell, which physicians could subsequently transplant into a patient to replace cells damaged by these same disease processes.

Scientists are gradually learning to direct the differentiation of pluripotent cell cultures into a specific type of cell, which can then be used as cellular models of human disease for drug discovery or toxicity studies. While it is not possible to predict the myriad ways that a basic understanding of stem cell differentiation may lead to new approaches for treating patients with cellular degenerative diseases, some avenues can be theorized. For example, in the case of Huntington's disease, a fatal neurodegenerative disorder, one could imagine that pluripotent cells derived from an embryo that carries Huntington's disease and differentiated into neurons in culture could be used to test drugs to delay or prevent degeneration.

Despite the incredible growth in knowledge that has occurred in stem cell research within the last couple of decades, investigators are just beginning to unravel the process of differentiation. Human pluripotent cell lines are an essential tool to understand this process and to facilitate the ultimate use of these cells in the clinic. To provide background on this fundamental topic, this article reviews the various potential sources and approaches that have been used to generate human pluripotent and multipotent cell lines, both of embryonic and non-embryonic origin.

Currently, at least six embryonic sources have been used to establish human pluripotent stem cell lines. All approaches involve isolation of viable cells during an early phase of development, followed by growth of these cells in appropriate culture medium. The various sources of these initial cell populations are discussed in brief below. It should be noted that the manipulation and use of embryonic tissues has raised a number of ethical issues.2,3 This article focuses on the scientific and technical issues associated with creating pluripotent cells, with the understanding that some of these techniques are currently subject to debates that extend beyond discussions of their scientific merits.

Figure 8.2. The Promise of Stem Cell ResearchStem cell research provides a useful tool for unraveling the molecular mechanisms that determine the differentiation fate of a pluripotent cell and for understanding the gene expression properties and epigenetic modifications essential to maintain the pluripotent state. In the future, this knowledge may be used to generate cells for transplantation therapies, whereby a specific cell population compromised by disease is replaced with new, functional cells. Differentiated derivatives of human pluripotent cells may also prove to be useful as models for understanding the biology of disease and developing new drugs, particularly when there is no animal model for the disease being studied. The greatest promise of stem cell research may lie in an area not yet imagined.

2008 Terese Winslow

Drawing upon twenty years of communal expertise with mouse ES cells,4 and on human inner cell mass culture conditions developed by Ariff Bongso and colleagues,5 James Thomson and colleagues at the University of Wisconsin generated the first hESC lines in 1998 using tissue from embryos fertilized in vitro.6 This method uses embryos generated for in vitro fertilization (IVF) that are no longer needed for reproductive purposes. During IVF, medical professionals usually produce more embryos than a couple attempting to start a family may need. Spare embryos are typically stored in a freezer to support possible future attempts for additional children if desired. It is estimated that there are approximately 400,000 such spare embryos worldwide.6 If these embryos are never used by the couple, they either remain in storage or are discarded as medical waste. Alternatively, these embryos can potentially be used to generate a hESC line.

To generate a hESC line, scientists begin with a donated blastocyst-stage embryo, at approximately five days after IVF (see Figure 8.3a). The blastocyst consists of approximately 150200 cells that form a hollow sphere of cells, the outer layer of which is called the trophectoderm. During normal development, the trophoblast becomes the placenta and umbilical cord. At one pole of this hollow sphere, 3050 cells form a cluster that is called the inner cell mass (ICM), which would give rise to the developing fetus. ICM cells are pluripotent, possessing the capacity to become any of the several hundred specialized cell types found in a developed human, with the exception of the placenta and umbilical cord.

Scientists remove the ICM from the donated blastocyst and place these cells into a specialized culture medium. In approximately one in five attempts, a hESC line begins to grow. Stem cells grown in such a manner can then be directed to differentiate into various lineages, including neural precursor cells,8 cardiomyocytes,9 and hematopoietic (blood forming) precursor cells.10

However, hESC lines are extremely difficult to grow in culture; the cells require highly specialized growth media that contain essential ingredients that are difficult to standardize. Yet the culture conditions are critical to maintain the cells' self-renewing and pluripotent properties. Culture requires the support of mouse or human cells, either directly as a "feeder" cell layer6,11,12 or indirectly as a source of conditioned medium in feeder-free culture systems.13 The feeder cells secrete important nutrients and otherwise support stem cell growth, but are treated so they cannot divide. Although the complete role of these feeder cells is not known, they promote stem cell growth, including detoxifying the culture medium and secreting proteins that participate in cell growth.14 hESC lines used to produce human cells for transplantation therapies may need to be propagated on a human feeder cell layer to reduce the risk of contamination by murine viruses or other proteins that may cause rejection. Thus, hESC lines often grow only under highly specific culture conditions, and the identification of ideal growth conditions presents a challenge regardless of the source of the hESCs.

Furthermore, human ES cell cultures must be expanded using an exacting protocol to avoid cell death and to control spontaneous differentiation. Since a limited number of laboratories in the United States are growing these cells, there is a shortage of people well-versed in the art and science of successful hESC culture. In the short term, challenges of working with these cells include developing robust culture conditions and protocols, understanding the molecular mechanisms that direct differentiation into specific cell types, and developing the infrastructure to advance this scientific opportunity. Once these challenges have been met, scientists will need to conduct transplantation studies in animal models (rodent and non-human primates) to demonstrate safety, effectiveness, and long-term benefit before stem cell therapies may enter clinical trials.

A second method for generating human pluripotent stem cell lines was published in 1998 by John Gearhart and coworkers at The Johns Hopkins Medical School.15

These researchers isolated specialized cells known as primordial germ cells (PGCs) from a 57-week-old embryo and placed these cells into culture (see Figure 8.3b). PGCs are destined to become either oocytes or sperm cells, depending on the sex of the developing embryo. The resulting cell lines are called embryonic germ cell lines, and they share many properties with ES cells. As with ES cells, however, PGCs present challenges with sustained growth in culture.16,17 Spontaneous differentiation, which hinders the isolation of pure clonal lines, is a particular issue. Therefore, the clinical application of these cells requires a more complete understanding of their derivation and maintenance in vitro.

Embryos that stop dividing after being fertilized in vitro are not preferentially selected for implantation in a woman undergoing fertility treatment. These embryos are typically either frozen for future use or discarded as medical waste. In 2006, scientists at the University of Newcastle, United Kingdom, generated hESC lines from IVF embryos that had stopped dividing.18 These scientists used similar methods as described under "Traditional hESC Line Generation" except that their source material was so-called "dead" IVF embryos (see Figure 8.3c). The human stem cells created using this technique behaved like pluripotent stem cells, including producing proteins critical for "stemness" and being able to produce cells from all three germ layers. It has been proposed that an IVF embryo can be considered dead when it ceases to divide.19 If one accepts this definition, such an embryo that "dies" from natural causes presumably cannot develop into a human being, thereby providing a source to derive human ES cells without destroying a living embryo.

Figure 8.3. Alternative Methods for Preparing Pluripotent Stem Cells

2008 Terese Winslow

Couples who have learned that they carry a genetic disorder sometimes use pre-implantation genetic diagnosis (PGD) and IVF to have a child that does not carry the disorder. PGD requires scientists to remove one cell from a very early IVF human embryo and test it for diseases known to be carried by the hopeful couple. Normally, embryos identified with genetic disorders are discarded as medical waste. However, Dr.Yuri Verlinsky and colleagues have capitalized on these embryos as a way to further our understanding of the diseases they carry (see Figure 8.3d) by deriving hESC lines from them.20 These stem cell lines can then be used to help scientists understand genetically-based disorders such as muscular dystrophy, Huntington's disease, thalessemia, Fanconi's anemia, Marfan syndrome, adrenoleukodystrophy, and neurofibromatosis.

In 2006, Dr. Robert Lanza and colleagues demonstrated that it is possible to remove a single cell from a pre-implantation mouse embryo and generate a mouse ES cell line.21 This work was based upon their experience with cleavage-stage mouse embryos. Later that same year, Dr. Lanza's laboratory reported that it had successfully established hESC lines (see Figure 8.3e) from single cells taken from pre-implantation human embryos.22 The human stem cells created using this technique behaved like pluripotent stem cells, including making proteins critical for "stemness" and producing cells from all three germ layers. Proponents of this technique suggest that since it requires only one embryonic cell, the remaining cells may yet be implanted in the womb and develop into a human being. Therefore, scientists could potentially derive human embryonic stem cells without having to destroy an embryo. However, ethical considerations make it uncertain whether scientists will ever test if the cells remaining after removal of a single cell can develop into a human being, at least in embryos that are not at risk for carrying a genetic disorder. Moreover, it is unclear whether the single cell used to generate a pluripotent stem cell line has the capacity to become a human being.

Parthenogenesis is the creation of an embryo without fertilizing the egg with a sperm, thus omitting the sperm's genetic contributions. To achieve this feat, scientists "trick" the egg into believing it is fertilized, so that it will begin to divide and form a blastocyst (see Figure 8.3f). In 2007, Dr. E.S. Revazova and colleagues reported that they successfully used parthenogenesis to derive hESCs.23 These stem cell lines, derived and grown using a human feeder cell layer, retained the genetic information of the egg donor and demonstrated characteristics of pluripotency. This technique may lead to the ability to generate tissue-matched cells for transplantation to treat women who are willing to provide their own egg cells.24 It also offers an alternate method for deriving tissue-matched hESCs that does not require destruction of a fertilized embryo.

Amniotic fluid surrounding the developing fetus contains cells shed by the fetus and is regularly collected from pregnant women during amniocentesis. In 2003, researchers identified a subset of cells in amniotic fluid that express Oct-4, a marker for pluripotent human stem cells that is expressed in ES cells and embryonic germ cells.25 Since then, investigators have shown that amniotic fluid stem cells can differentiate into cells of all three embryonic germ layers and that these cells do not form tumors in vivo.26,27

For example, Anthony Atala and colleagues at the Wake Forest University have recently generated non-embryonic stem cell lines from cells found in human and rat amniotic fluid.27 They named these cells amniotic fluid-derived stem cells (AFS). Experiments demonstrate that AFS can produce cells that originate from each of the three embryonic germ layers, and the self-renewing cells maintained the normal number of chromosomes after a prolonged period in culture. However, undifferentiated AFS did not produce all of the proteins expected of pluripotent cells, and they were not capable of forming a teratoma. The scientists developed in vitro conditions that enabled AFS to produce nerve cells, liver cells, and bone-forming cells. AFS-derived human nerve cells could make proteins typical of specialized nerve cells and were able to integrate into a mouse brain and survive for at least two months. Cultured AFS-derived human liver cells secreted urea and made proteins characteristic of normal human liver cells. Cultured AFS-derived human bone cells made proteins expected of human bone cells and formed bone in mice when seeded onto scaffolds and implanted under the mouse's skin. Although scientists do not yet know how many different cell types AFS can generate, AFS may one day allow researchers to establish a bank of cells for transplantation into humans.

An alternative to searching for an existing population of stem cells is to create a new one from a population of non-pluripotent cells. This strategy, which may or may not involve the creation of an embryo, is known as "reprogramming." This section will summarize reprogramming approaches, including several recent breakthroughs in the field..

In SCNT (see Figure 8.3g), human oocytes (eggs) are collected from a volunteer donor who has taken drugs that stimulate the production of more than one oocyte during the menstrual cycle. Scientists then remove the nucleus from the donated oocyte and replace it with the nucleus from a somatic cell, a differentiated adult cell from elsewhere in the body. The oocyte with the newly-transferred nucleus is then stimulated to develop. The oocyte may develop only if the transplanted nucleus is returned to the pluripotent state by factors present in the oocyte cytoplasm. This alteration in the state of the mature nucleus is called nuclear reprogramming. When development progresses to the blastocyst stage, the ICM is removed and placed into culture in an attempt to establish a pluripotent stem cell line. To date, the technique has been successfully demonstrated in two primates: macaque monkeys28 and humans.29

However, successful SCNT creates an embryo-like entity, thereby raising the ethical issues that confront the use of spare IVF embryos. However, pluripotent cell lines created by embryos generated by SCNT offer several advantages over ES cells. First, the nuclear genes of such a pluripotent cell line will be identical to the genes in the donor nucleus. If the nucleus comes from a cell that carries a mutation underlying a human genetic disease such as Huntington's disease, then all cells derived from the pluripotent cell line will carry this mutation. In this case, the SCNT procedure would enable the development of cellular models of human genetic disease that can inform our understanding of the biology of disease and facilitate development of drugs to slow or halt disease progression. Alternatively, if the cell providing the donor nucleus comes from a specific patient, all cells derived from the resulting pluripotent cell line will be genetically matched to the patient with respect to the nuclear genome. If these cells were used in transplantation therapy, the likelihood that the patient's immune system would recognize the transplanted cells as foreign and initiate tissue rejection would be reduced. However, because mitochondria also contain DNA, the donor oocyte will be the source of the mitochondrial genome, which is likely to carry mitochondrial gene differences from the patient which may still lead to tissue rejection.

A technique reported in 2007 by Dr. Kevin Eggan and colleagues at Harvard University may expand scientists' options when trying to "reprogram" an adult cell's DNA30. Previously, successful SCNT relied upon the use of an unfertilized egg. Now, the Harvard scientists have demonstrated that by using a drug to stop cell division in a fertilized mouse egg (zygote) during mitosis, they can successfully reprogram an adult mouse skin cell by taking advantage of the "reprogramming factors" that are active in the zygote at mitosis. They removed the chromosomes from the single-celled zygote's nucleus and replaced them with the adult donor cell's chromosomes (see Figure 8.3h). The active reprogramming factors present in the zygote turned genes on and off in the adult donor chromosomes, to make them behave like the chromosomes of a normally fertilized zygote. After the zygote was stimulated to divide, the cloned mouse embryo developed to the blastocyst stage, and the scientists were able to harvest embryonic stem cells from the resulting blastocyst. When the scientists applied their new method to abnormal mouse zygotes, they succeeded at reprogramming adult mouse skin cells and harvesting stem cells. If this technique can be repeated with abnormal human zygotes created in excess after IVF procedures, scientists could use them for research instead of discarding them as medical waste.

Altered nuclear transfer is a variation on standard SCNT that proposes to create patient-specific stem cells without destroying an embryo. In ANT, scientists turn off a gene needed for implantation in the uterus (Cdx2) in the patient cell nucleus before it is transferred into the donor egg (see Figure 8.3i). In 2006, Dr. Rudolph Jaenisch and colleagues at MIT demonstrated that ANT can be carried out in mice.31 Mouse ANT entities whose Cdx2 gene is switched off are unable to implant in the uterus and do not survive to birth. Although ANT has been used to create viable stem cell lines capable of producing almost all cell types, the authors point out that this technique must still be tested with monkey and human embryos. Moreover, the manipulation needed to control Cdx2 expression introduces another logistical hurdle that may complicate the use of ANT to derive embryonic stem cells. Proponents of ANT, such as William Hurlbut of the Stanford University Medical Center, suggest that the entity created by ANT is not a true embryo because it cannot implant in the uterus.32, 33 However, the technique is highly controversial, and its ethical implications remain a source of current debate.4,32

In 2005, Kevin Eggan and colleagues at Harvard University reported that they had fused cultured adult human skin cells with hESCs (see Figure 8.3j).36 The resulting "hybrid" cells featured many characteristics of hESCs, including a similar manner of growth and division and the manufacture of proteins typically produced by hESCs. Some factor(s) within the hESCs enabled them to "reprogram" the adult skin cells to behave as hESCs. However, these cells raised a significant technical barrier to clinical use. Because fused cells are tetraploid (they contain four copies of the cellular DNA rather than the normal two copies), scientists would need to develop a method to remove the extra DNA without eliminating their hESC-like properties. The fusion method serves as a useful model system for studying how stem cells "reprogram" adult cells to have properties of pluripotent cells. However, if the reprogramming technique could be carried out without the fusion strategy, a powerful avenue for creating patient-specific stem cells without using human eggs could be developed.

In 2007, two independent research groups published manuscripts that described successful genetic reprogramming of human adult somatic cells into pluripotent human stem cells.34,35 Although some technical limitations remain, this strategy suggests a promising new avenue for generating pluripotent cell lines that can inform drug development, models of disease, and ultimately, transplantation medicine. These experiments, which are discussed below, were breakthroughs because they used adult somatic cells to create pluripotent stem cells that featured hallmarks of ES cells.

In 2006, Shinya Yamanaka and colleagues at Kyoto University reported that they could use a retroviral expression vector to introduce four important stem cell factors into adult mouse cells and reprogram them to behave like ES cells (see Figure 8.3k).37 They called the reprogrammed cells "iPSCs," for induced pluripotent stem cells. However, iPSCs produced using the original technique failed to produce sperm and egg cells when injected into an early mouse blastocyst and did not make certain critical DNA changes. These researchers then modified the technique to select for iPSCs that can produce sperm and eggs,38 results that have since been reproduced by Rudolph Jaenisch and colleagues at the Massachusetts Institute of Technology (MIT).39

In addition, the MIT scientists determined that iPSCs DNA is modified in a manner similar to ES cells, and important stem cell genes are expressed at similar levels. They also demonstrated that iPSCs injected into an early mouse blastocyst can produce all cell types within the developing embryo, and such embryos can complete gestation and are born alive.

Once these research advances were made in mice, they suggested that similar techniques might be used to reprogram adult human cells. In 2007, Yamanaka and coworkers reported that introducing the same four genetic factors that reprogrammed the mouse cells into adult human dermal fibroblasts reprogrammed the cells into human iPSCs.35 These iPSCs were similar to human ES cells in numerous ways, including morphology, proliferative capacity, expression of cell surface antigens, and gene expression. Moreover, the cells could differentiate into cell types from the three embryonic germ layers both in vitro and in teratoma assays. Concurrent with the Yamanaka report, James Thomson and coworkers at the University of Wisconsin published a separate manuscript that detailed the creation of human iPSCs through somatic cell reprogramming using four genetic factors (two of which were in common with the Yamanaka report).34 The cells generated by the Thomson group met all defining criteria for ES cells, with the exception that they were not derived from embryos.

These breakthroughs have spurred interest in the field of iPSCs research. In early 2008, investigators at the Massachusetts General Hospital40 and the University of California, Los Angeles41 reported generating reprogrammed cells. As scientists explore the mechanisms that govern reprogramming, it is anticipated that more reports will be forthcoming in this emerging area. Although these reprogramming methods require the use of a virus, non-viral strategies may also be possible in the future. In any case, these approaches have created powerful new tools to enable the "dedifferentation" of cells that scientists had previously believed to be terminally differentiated.42,43

Although further study is warranted to determine if iPS and ES cells differ in clinically significant ways, these breakthrough reports suggest that reprogramming is a promising strategy for future clinical applications. Induced pluripotent cells offer the obvious advantage that they are not derived from embryonic tissues, thereby circumventing the ethical issues that surround use of these materials. Successful reprogramming of adult somatic cells could also lead to the development of stem cell lines from patients who suffer from genetically-based diseases, such as Huntington's Disease, spinal muscular atrophy, muscular dystrophy, and thalessemia. These lines would be invaluable research tools to understand the mechanisms of these diseases and to test potential drug treatments. Additionally, reprogrammed cells could potentially be used to repair damaged tissues; patient-specific cell lines could greatly reduce the concerns of immune rejection that are prevalent with many transplantation strategies.

However, several technical hurdles must be overcome before iPSCs can be used in humans. For example, in preliminary experiments with mice, the virus used to introduce the stem cell factors sometimes caused cancers.37 The viral vectors used in these experiments will have to be selected carefully and tested fully to verify that they do not integrate into the genome, thereby harboring the potential to introduce genetic mutations at their site of insertion. This represents a significant concern that must be addressed before the technique can lead to useful treatments for humans. However, this strategy identifies a method for creating pluripotent stem cells that, together with studies of other types of pluripotent stem cells, will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

Stem cell research is a rapidly evolving field, and researchers continue to isolate new pluripotent cells and create additional cell lines. This section briefly reviews other sources of pluripotent cells and the implications that their discovery may have on future research.

Epiblast Cells. While rodent and human ES cells are pluripotent, they maintain their respective pluripotencies through different molecular signaling pathways. It is not known why these differences exist. Recently, several research groups have reported the generation of stable, pluripotent cell lines from mouse and rat epiblast, a tissue of the post-implantation embryo that ultimately generates the embryo proper.44,45 These cells are distinct from mouse ES cells in terms of the signals that control their differentiation. However, the cells share patterns of gene expression and signaling responses with human ES cells. The establishment of epiblast cell lines can therefore provide insight into the distinctions between pluripotent cells from different species and illuminate ways that pluripotent cells pursue distinct fates during early development.

Existing Adult Stem Cells. As has been discussed in other chapters, numerous types of precursor cells have been isolated in adult tissues.46 Although these cells tend to be relatively rare and are dispersed throughout the tissues, they hold great potential for clinical application and tissue engineering. For example, tissues created using stem cells harvested from an adult patient could theoretically be used clinically in that patient without engendering an immune response. Moreover, the use of adult stem cells avoids the ethical concerns associated with the use of ES cells. In addition, adult-derived stem cells do not spontaneously differentiate as do ES cells, thus eliminating the formation of teratomas often seen with implantation of ES cells. The potential of adult stem cells for regenerative medicine is great; it is likely that these various cells will find clinical application in the upcoming decades.

Although the recent advances in reprogramming of adult somatic cells has generated a wave of interest in the scientific community, these cell lines will not likely replace hESC lines as tools for research and discovery. Rather, both categories of cells will find unique uses in the study of stem cell biology and the development and evaluation of therapeutic strategies. Pluripotent cells offer a number of potential clinical applications, especially for diseases with a genetic basis. However, researchers are just beginning to unlock the many factors that govern the cells' growth and differentiation. As scientists make strides toward understanding how these cells can be manipulated, additional applications, approaches, and techniques will likely emerge. As such, pluripotent cells will play a pivotal role in future research into the biology of development and the treatment of disease.

Chapter7|Table of Contents|Chapter9

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Alternate Methods for Preparing Pluripotent Stem Cells ...