14 hours ago Growing stem cells in conditions free of animal material makes them safe for use in humans. Credit: Eraxion/iStock/Thinkstock

Human stem cells produced through genetic reprogramming are beset by safety concerns because current techniques alter the DNA of the stem cells and use material from animals to grow them. Now, A*STAR researchers have developed an efficient approach that produces safe, patient-specific human stem cells.

Human induced pluripotent stem cells have the potential to treat a number of diseases without the ethical issues associated with embryonic stem cells. Pluripotent stem cells can be produced from adult cells by introducing genes that reprogram them. Typically, the stem cells are grown on a layer of mouse cells in solutions (known as media) that contain animal proteinsand therefore, potentially may also carry disease. For such stem cells to be safe for use in humans, they need to be grown in 'xeno-free' conditions, which are devoid of material from other animals.

Andrew Wan and Hong Fang Lu at the A*STAR Institute of Bioengineering and Nanotechnology in Singapore and colleagues set out to develop a new xeno-free system. The researchers carried out the genetic reprogramming of cells on an artificially produced protein substrate rather than mouse cells. They also used media that contained no animal components. The result was more efficient reprogramming than seen with conventional approaches.

"A xeno-free system will eliminate the risk of disease transmission from other species, which is important for regulatory approval," explains Wan. "Yet there have been few studies on cell reprogramming under totally xeno-free conditions."

The researchers went one step further by addressing the problem of cells acquiring alterations to their DNA during reprogramming.

"Incorporation of transgenes into the genome of the cell poses another safety issue, risking unwanted genetic alterations," explains Lu. "In our work, the transgenes were introduced to initiate the reprogramming, but after this they were removed from the cell, leading to transgene-free stem cells."

The researchers demonstrated that after genetic reprogramming and the removal of the added genes, the stem cells could still develop into different cells types. They were even able to induce them to form dopaminergic neurons, the type that degenerates in Parkinson's disease. The conditions in which the stem cells were grown mean that they are suitable for clinical use and can be derived from a patient's own cells, ensuring complete compatibility.

"Regulatory approval for clinical application of stem cells largely depends on the conditions in which the stem cells are derived," says Wan. "We present a workable protocol for the reprogramming of fibroblasts to stem cells that minimizes any potential safety risks."

Explore further: Discovery may make it easier to develop life-saving stem cells

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Animal-free reprogramming of adult cells improves safety

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Newswise Hematopoietic stem cells (HSCs) give rise to all other blood cell types, but their development and how their fate is determined has long remained a mystery. In a paper published online this week in Nature, researchers at the University of California, San Diego School of Medicine elaborate upon a crucial signaling pathway and the role of key proteins, which may help clear the way to generate HSCs from human pluripotent precursors, similar to advances with other kinds of tissue stem cells.

Principal investigator David Traver, PhD, professor in the Department of Cellular and Molecular Medicine, and colleagues focused on the Notch signaling pathway, a system found in all animals and known to be critical to the generation of HSCs in vertebrates. Notch signaling between emitting and receiving cells is key to establishing HSC fate during development, said Traver. What has not been known is where, when and how Notch signal transduction is mediated.

Traver and colleagues discovered that the Notch signal is transduced into HSC precursor cells from signal emitting cells in the somite embryologic tissues that eventually contribute to development of major body structures, such as skeleton, muscle and connective tissues much earlier in the process than previously anticipated.

More specifically, they found that JAM proteins, best known for helping maintain tight junctions between endothelial cells to prevent vascular leakage, were key mediators of Notch signaling. When the researchers caused loss of function in JAM proteins in a zebrafish model, Notch signaling and HSCs were also lost. When they enforced Notch signaling through other means, HSC development was rescued.

To date, it has not been possible to generate HSCs de novo from human pluripotent precursors, like induced pluripotent stem cells, said Traver. This has been due in part to a lack of understanding of the complete set of factors that the embryo uses to make HSCs in vivo. It has also likely been due to not knowing in what order each required factor is needed.

Our studies demonstrate that Notch signaling is required much earlier than previously thought. In fact, it may be one of the earliest determinants of HSC fate. This finding strongly suggests that in vitro approaches to instruct HSC fate from induced pluripotent stem cells must focus on the Notch pathway at early time-points in the process. Our findings have also shown that JAM proteins serve as a sort of co-receptor for Notch signaling in that they are required to maintain close contact between signal-emitting and signal-receiving cells to permit strong activation of Notch in the precursors of HSCs.

The findings may have far-reaching implications for eventual development of hematopoietic stem cell-based therapies for diseases like leukemia and congenital blood disorders. Currently, it is not possible to create HSCs from differentiation of embryonic stem cells or induced pluripotent stem cells pluripotent cells artificially derived from non-pluripotent cells, such as skin cells that are being used in other therapeutic research efforts.

Co-authors include Isao Kobayashi, Jingjing Kobayashi-Sun, Albert D. Kim and Claire Pouget, UC San Diego Department of Cellular and Molecular Medicine; Naonobu Fujita, UC San Diego Section of Cell and Developmental Biology; and Toshio Suda, Keio University, Japan.

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New Blood: Tracing the Beginnings of Hematopoietic Stem Cells

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Newswise During pregnancy, certain hormones trigger specialized mammary stem cells to create milk-producing cells essential to lactation. Scientists at the University of California, San Diego School of Medicine and Moores Cancer Center have found that mammary stem cells associated with the pregnant mammary gland are related to stem cells found in breast cancer.

Writing in the August 11, 2014 issue of Developmental Cell, David A. Cheresh, PhD, Distinguished Professor of Pathology and vice-chair for research and development, Jay Desgrosellier, PhD, assistant professor of pathology and colleagues specifically identified a key molecular pathway associated with aggressive breast cancers that is also required for mammary stem cells to promote lactation development during pregnancy.

By understanding a fundamental mechanism of mammary gland development during pregnancy, we have gained a rare insight into how aggressive breast cancer might be treated, said Cheresh. This pathway can be exploited. Certain drugs are known to disrupt this pathway and may interfere with the process of breast cancer progression.

During pregnancy, a new mammary stem cell population arises, distinct from those involved in development and maintenance of the non-pregnant gland. These stem cells remodel the breasts and lactating glands in preparation for feeding the newborn child. Normally, these stem cells contribute only to early remodeling events and are switched off by the time milk production begins.

The researchers found, however, that signals regulating stem cell activation during pregnancy appear to be hijacked by cancer cells to produce faster-growing, more aggressive tumors. This normal pathway ends up contributing to the progression of cancer, said Desgrosellier, first author of the study.

A connection between pregnancy and breast cancer has long been known. But the association between pregnancy and breast cancer risk is complex. While having a child reduces a womans risk of developing breast cancer later in life, there is also an increased short-term risk for the development of a highly aggressive form of breast cancer following each pregnancy. The current study suggests that molecules important for stem cell behavior during pregnancy may contribute to these more aggressive pregnancy-associated breast cancers, a possibility the researchers plan to investigate further.

The authors are quick to point out that their findings should not be interpreted as a reason to avoid pregnancy. The signaling pathway usurped by cancer cells is not the cause of breast cancer. Rather, they said, it may worsen or accelerate a cancer caused by other factors, such as an underlying mutation or genetic predisposition.

Our work doesnt speak to the actual cause of cancer. Rather, it explains what can happen once cancer has been initiated, said Cheresh. Heres an analogy: To get cancer, you first have to start with an oncogene, a gene that carries a mutation and has the potential to initiate cancer. Think of the oncogene as turning on a cars ignition. The signaling pathway exploited by cancer cells is like applying gas. It gets the car moving, but it means nothing if the oncogene hasnt first started the process.

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How Breast Cancer Usurps the Powers of Mammary Stem Cells

During pregnancy, certain hormones trigger specialized mammary stem cells to create milk-producing cells essential to lactation. Scientists at the University of California, San Diego School of Medicine and Moores Cancer Center have found that mammary stem cells associated with the pregnant mammary gland are related to stem cells found in breast cancer.

Writing in the August 11, 2014 issue of Developmental Cell, David A. Cheresh, PhD, Distinguished Professor of Pathology and vice-chair for research and development, Jay Desgrosellier, PhD, assistant professor of pathology and colleagues specifically identified a key molecular pathway associated with aggressive breast cancers that is also required for mammary stem cells to promote lactation development during pregnancy.

"By understanding a fundamental mechanism of mammary gland development during pregnancy, we have gained a rare insight into how aggressive breast cancer might be treated," said Cheresh. "This pathway can be exploited. Certain drugs are known to disrupt this pathway and may interfere with the process of breast cancer progression."

During pregnancy, a new mammary stem cell population arises, distinct from those involved in development and maintenance of the non-pregnant gland. These stem cells remodel the breasts and lactating glands in preparation for feeding the newborn child. Normally, these stem cells contribute only to early remodeling events and are switched off by the time milk production begins.

The researchers found, however, that signals regulating stem cell activation during pregnancy appear to be hijacked by cancer cells to produce faster-growing, more aggressive tumors. "This normal pathway ends up contributing to the progression of cancer," said Desgrosellier, first author of the study.

A connection between pregnancy and breast cancer has long been known. But the association between pregnancy and breast cancer risk is complex. While having a child reduces a woman's risk of developing breast cancer later in life, there is also an increased short-term risk for the development of a highly aggressive form of breast cancer following each pregnancy. The current study suggests that molecules important for stem cell behavior during pregnancy may contribute to these more aggressive pregnancy-associated breast cancers, a possibility the researchers plan to investigate further.

The authors are quick to point out that their findings should not be interpreted as a reason to avoid pregnancy. The signaling pathway usurped by cancer cells is not the cause of breast cancer. Rather, they said, it may worsen or accelerate a cancer caused by other factors, such as an underlying mutation or genetic predisposition.

"Our work doesn't speak to the actual cause of cancer. Rather, it explains what can happen once cancer has been initiated," said Cheresh. "Here's an analogy: To get cancer, you first have to start with an oncogene, a gene that carries a mutation and has the potential to initiate cancer. Think of the oncogene as turning on a car's ignition. The signaling pathway exploited by cancer cells is like applying gas. It gets the car moving, but it means nothing if the oncogene hasn't first started the process."

The researchers focused on a family of cell surface receptor proteins called integrins that act as key communications conduits, ultimately zeroing in on the role of one member of this family called beta-3 integrin. Also known as CD61, it was already linked to metastasis and resistance to cancer drugs.

Cheresh noted that CD61 represents a good marker for the incriminated signaling pathway involved in both mammary development during pregnancy and cancer. It's easily detected and could be used to both diagnose and treat breast cancer cases. "Detecting CD61 might help doctors determine what kind of therapeutic approach to use, knowing that they might be dealing with a more aggressive yet treatable form of breast cancer. For example, there are existing drugs that block CD61 signaling, which might be another potential aspect of treatment."

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How breast cancer usurps powers of mammary stem cells