Researcher Dhruv Sareens own stem cells are now orbiting the Earth. The mission? To test whether theyll grow better in zero gravity.

Scientists at Cedars-Sinai Medical Center in Los Angeles are trying to find new ways to produce huge batches of a type of stem cell that can generate nearly any other type of cell in the body and potentially be used to make treatments for many diseases. The cells arrived over the weekend at the International Space Station on a supply ship.

I dont think I would be able to pay whatever it costs now" to take a private ride to space, Sareen said. At least a part of me in cells can go up!"

The experiment is the latest research project that involves shooting stem cells into space. Some, like this one, aim to overcome the terrestrial difficulty of mass producing the cells. Others explore how space travel impacts the cells in the body. And some help better understand diseases such as cancer.

By pushing the boundaries like this, its knowledge and its science and its learning, said Clive Svendsen, executive director of Cedars-Sinais Regenerative Medicine Institute.

Six earlier projects from the U.S., China and Italy sent up various types of stem cells including his teams study of the effects of microgravity on cell-level heart function, said Dr. Joseph Wu of Stanford University, who directs the Stanford Cardiovascular Institute. Wu helped coordinate a series of programs on space-based stem cell research last year.

Earthly applications of much of this research may be a little ways off.

At this point, the only stem cell-based products approved by the Food and Drug Administration contain blood-forming stem cells from umbilical cord blood for patients with blood disorders such as certain cases of lymphoma. There are no approved therapies using the kind of stem cells being sent to space or others derived from them, said Jeffrey Millman, a biomedical engineering expert at Washington University in St. Louis.

But clinical trials underway involving stem cells target conditions such as macular degeneration, Parkinsons disease and heart attack damage. And Millman is involved in research that could lead to a new approach for treating Type 1 diabetes.

Scientists see great promise in stem cells.

THE GRAVITY DILEMMA

That promise is tempered by a frustrating earthly problem: The planets gravity makes it tough to grow the vast quantities of cells necessary for future therapies that may require more than a billion per patient.

With current technology right now, even if the FDA instantly approved any of these therapies, we dont have the capacity to manufacture what's needed, Millman said.

The issue? In large bioreactors, the cells need to be stirred vigorously or they clump or fall to the bottom of the tank, Millman said. The stress can cause most cells to die.

In zero G, theres no force on the cells, so they can just grow in a different way, Svendsen said.

The Cedars-Sinai team has sent up what are called induced pluripotent stem cells. Many scientists consider them the perfect starting materials for all sorts of personalized, cell-based treatments. They carry a patients own DNA, and their versatility makes them similar to embryonic stem cells, only they are reprogrammed from adults' skin or blood cells.

For their experiment, which is being funded by NASA, a shoebox-sized container holds bags filled with spheres of cells and all of the pumps and solutions needed to keep them alive for four weeks. The cargo will also include neural stem cells originating from Svendsen. The scientists used stem cells derived from their own white blood cells because it was easy for them to give consent.

They will run the experiment remotely with a box of cells on Earth for comparison. They'll get the space experiment back in five weeks or so, when it returns in the same SpaceX capsule.

The work is designed to pave the way for more NASA-funded research. If they are able to figure out how to make billions of cells in orbit, Svendsen said, the impact could be huge.

A HIGH-FLYING FUTURE

During the same cargo launch, researchers from the University of California, San Diego, sent blood stem cells to the space station, a repeat of an experiment they did last year. They want to find out if low Earth orbit induces faster aging in the cells, leading to problems that set the stage for precancerous changes. One goal is to protect astronauts' health.

Afshin Beheshti, a researcher at NASA Ames Research Center, said scientists are just beginning to understand some of the risks of space travel.

Theres more unknowns in space than there are knowns," he said. Any new type of experiment is going to shed light on how the body responds to the space environment.

Ultimately, Beheshti said, the research should yield more than practical, earthly solutions like new medicines. It will also help with far-off human aspirations, like living on other planets.

The Associated Press Health and Science Department receives support from the Howard Hughes Medical Institutes Department of Science Education. The AP is solely responsible for all content.

Excerpt from:
High-flying experiment: Do stem cells grow better in space? - ABC News

Mesenchymal stem cell isolation and expansion

Bone marrow-derived MSCs were isolated from young (6 weeks) and old (1824 months) C57 black male mice using established techniques42,43 under a protocol approved by the Johns Hopkins University Animal Care and Use Committee. Briefly, immediately following euthanasia, whole bone marrow was flushed out from the bilateral tibias and femurs. After washing by centrifugation at 400g for 10min, cells were plated at 5 106 viable cells per ml. The culture was kept in humidified 5% CO2 incubator at 37C for 72h, when non-adherent cells were removed by changing the media.

All MSC preparations were evaluated using flow cytometry with PE or FITC-conjugated antibodies against murine Sca-1 (1:200; BioLegend 122507), CD31 (1:200; Fisher Scientific BDB554473), CD34 (1:100; eBioscience 14-0341-82), CD44 (1:100; BioLegend 103007), CD45 (1:100; BioLegend 103105), and IgG (1:100; BioLegend 400607) performed on BD LSRII (Becton Dickinson) using DIVA software. At least 10000 events were collected. FlowJo software was used to analyze and create the histograms.

Assessment for osteogenic and adipogenic differentiation was performed using established techniques43. Briefly, to induce osteogenic differentiation, old and young MSCs were seeded into 6-well plates at 1.3 104 cells/well. After 24h the media was replaced with osteogenic differentiation medium containing Iscoves medium supplemented with 100nM dexamethasone, 10mM beta-glycerophosphate, 50 M ascorbic acid, and 1% antibiotic/antimycotic. Cells were maintained in induction media with media changes every 2 days. After 14 days cells fixed in 10% formalin for 15min and calcium deposition was assessed using von Kossa staining. Calcium deposition was then quantified using a colorimetric calcium assay (Calcium CPC Liquicolour Kit StanBio, Boerne, TX) according to the manufacturers instructions. To induce adipogenic differentiation, old and young MSCs were seeded in 6-well plates at 2 105 cells/well. When confluent the media was replaced with adipogenic induction medium containing DMEM-HG, 10% FBS, 5% rabbit serum, 1uM dexamethasone, 10g/mL insulin, 200 M indomethacin, 500 M isobutylmethylxanthine (IBMX), and antibiotic/antimycotic for 3 days followed by exposure to followed by exposure to adipogenic maintenance medium (DMEM-HG, 10% FBS, insulin 10g/ml and P/S) for 3 days. After 3 cycles of induction and maintenance exposure cells were rinsed with PBS and fixed in 10% formalin for 10min. The cells were then stained with Oil Red O to assess for lipid droplets. After imaging Oil Red O extraction was performed using 100% isopropanol. Extract samples were transferred to a 96-well plate and absorbance readings were taken at 490nm to quantify extracted Oil Red O.

Confirmed MSCs were expanded in culture in media prepared by combining 490ml Medium 200 PRF (Gibco Invitrogen, Carlsbad, CA), a standard basal medium intended for culture of large vessel human endothelial cells, with 10ml Low Serum Growth Supplement (LSGS; Gibco Invitrogen). The final preparation contained 2% fetal bovine serum (FBS), 3ng/ml basic fibroblast growth factor (bFGF), 10ng/ml human epidermal growth factor, 10g/ml heparin, and 1g/ml hydrocortisone. Cells were incubated under standard conditions (5% CO2 and 37C). Expanded MSCs at low passage numbers (P2-P5) were used for the experiments. In the event frozen cells were used, they were thawed and grown for one passage prior to use in the experiments.

To prevent cell-cell interaction and assess only paracrine-mediated effects (i.e. those resulting from release of soluble factors), angiogenesis experiments were performed using bioreactor tubes (BT) constructed with CellMax semi-permeable polysulfone membrane tubing (Spectrum Labs, Rancho Dominguez, CA). These allowed the free diffusion of soluble proteins and other molecules released by the cells up to a 500kDa molecular weight cut-off, but not of the cells themselves. To load BTs, MSCs were trypsinized and suspended in Medium 200 PRF without LSGS supplementation (i.e. media devoid of stimulatory growth factors). MSCs were counted using a Scepter automated cell counter (Millipore, Billerica, MA), which had been previously standardized for accuracy. The desired number of MSCs was spun down and resuspended to a total volume of 100 ul that was injected into the BTs using a 0.5mL syringe. To compare paracrine-mediated angiogenesis by old and young MSCs, BTs were loaded with either 105 old or 105 young MSCs. Once cell injection was complete, the tubes were heat-sealed at both ends and the MSC-loaded tubes, fully submerged in media, were grown at standard culture conditions (37C, 5% CO2) for 7 days (Fig. 3a).

ELISA assays were performed to measure paracrine factor (PF) production by the MSCs contained within the BTs grown in culture. Tubes loaded with 2 105 MSCs were submerged in 5mL of alpha-MEM basal medium (Stemcell Technologies, Tukwila, WA) supplemented with 20% FBS (Gibco Invitrogen, Carlsbad, CA) in a 6-well plate. At day 7, conditioned media was collected from each well, spun down for 1min to pellet any debris, and then flash frozen at 80C. Conditioned media samples were assessed for the concentrations of vascular endothelial growth factor (VEGF), stromal derived factor-1 (SDF1) and insulin-like growth factor-1 (IGF1) by ELISA (Quantikine, R&D Systems, Minneapolis, MN) according to the manufacturers instructions.

BTs were removed at day 7 and placed in separate wells of a 6-well plate containing human umbilical vein endothelial cells (HUVECs)44. Briefly, 105 HUVECs (Gibco Invitrogen, Carlsbad, CA) suspended in Medium 200PRF were plated per well in Geltrex (Gibco Invitrogen) coated 6-well plates. Negative control wells received a bioreactor loaded with un-supplemented Medium 200PRF only (i.e. no cells). Positive control wells were plated with 105 HUVECs suspended in 1mL of Medium 200PRF supplemented with LSGS, which is known to induce HUVEC tubule formation. After 18h at standard culture conditions (37C, 5% CO2), the wells were imaged to allow quantitative analysis of the resultant HUVEC tubule network. Images were taken in the center of each well and in all four quadrants at pre-determined locations (5 pictures total), at 100x magnification. The total length of the tubule networks captured in the images of each well was measured using ImageJ software. To allow for comparisons between experiments, the total length of the tubule network in each well was normalized to the average length of the tubule network in the negative control wells, and reported as a normalized ratio.

To assess the effect of young MSC-generated PFs on PF-mediated angiogenesis by old MSCs, BTs were prepared as described above containing either 105 young or 105 old MSCs. Two BTs were placed together in a 6-well plate in 5mL MSC media and incubated for 7 days at standard culture conditions (Fig. 3b) using a BT containing old MSCs paired with either a separate BT with other old MSCs (control) or a separate BT with young MSCs. After 7 days the tubes were removed, washed with un-supplemented Medium 200 PRF, and then used separately in the HUVEC assay as described above. After the HUVEC assay was complete (18h) the BTs were placed in separate wells of 6-well plates and grown in culture for 7 additional days with collection of conditioned media for PF release.

Replicates of 105 old MSCs were cultured separately, or in co-culture with young MSCs, for 7 days using a 0.4m Transwell system in 6-well plates (Corning), which allow transfer of soluble paracrine factors released by the cells, but not of the cells themselves. Following RNA purification, library preparation, amplification, and Illumina sequencing, the open source Galaxy pipeline was used for data processing and analyses. After alignment of raw sequencing reads to the UCSC mm10 genome using HISAT2, transcript assembly, alignment quantification, count normalization, and differential expression analyses were conducted with StringTie, featureCounts, DESeq2, and Genesis. Quantitative PCR (KAPA SYBR FAST One-Step qRT-PCR, Wilmington, MA) was used to validate 24 transcripts identified by RNA sequencing. Target genes were selected based on their presence in significantly regulated pathways and quantified relative to 18S ribosomal RNA using the 2Ct method45.

To validate the results of the RNA sequencing and RT-PCR results, a functional autophagy assay was performed to assess relative autophagy between old, young, and rejuvenated old MSCs. Old, young and rejuvenated cells were cultured (or co-cultured, in the case of rejuvenated cells) for 7 days in 6-well plates (105 cells per well). On Day 8, cells were trypsinzed, counted and 104 cells were transferred to each well of a 96-well black plate with clear bottom and incubated for 6h. The Autophagy Assay Kit (Sigma Aldrich, St. Louis, MO) measures autophagy using a proprietary fluorescent autophagosome marker in a microplate reader (ex=360; em=520nm). Three separate experiments were performed in triplicate each for each condition. To account for possible differences introduced by counting cells, results for each cell type were normalized based on absorbance (450nm) of a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc. Rockville, MD).

Data are reported as mean standard error of the mean (SEM) unless otherwise indicated. Comparisons between groups for the HUVEC experiments were performed using the permutation test. For the PF ELISA data, groups were compared using the MannWhitney test. The autophagy assay and rt-PCR results were assessed using two-tailed t tests. For these experiments a p-value < 0.05 was deemed significant. In the RNA sequencing differential expression analysis, a false discovery rate (FDR) of <0.05 was considered significant.

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Paracrine-mediated rejuvenation of aged mesenchymal stem cells is associated with downregulation of the autophagy-lysosomal pathway | npj Aging -...

For the past seven weeks, four-year-old Minh Nguyen has spent most of her time in hospital, receiving regular blood transfusions because her doctors have not been able to find acompatible stem cell donor.

Minh was diagnosed with bone marrow aplasia, arare blood disease that affects her bone marrow's ability to generate white blood cells. She hasspent the last three weeks in hospital full-time.

"It's been hard for her because she doesn't have the life of a four-year-oldchild anymore," said Minh's mother, Diem Nguyen.

Nguyen was speaking at an event organized for Minh at a Montreal restaurant Saturday, where dozens of people with similar genetic makeups to Minh showed up to volunteer as potential stem cell donors.

There is an international registry of 25millionpeople who have provided consent and genetic information in order to donate stem cells, which come from the bone marrow of a healthy adult.

But about 70 per cent of those on the registry are white, putting donor recipients of from other ethnic backgroundsat a significant disadvantage.

Before Saturday's event, Minh's mother described her daughter's chances as one in a million.

Marie-Cindel Surprenant says she felt a personal connection to Minh Nguyen's story when learned about it from a colleague.

"I think that if somehow I can help her, I'll do it, so I think it's a good purpose in my life," she said.

Surprenant is half-Asian, half-white, like Minh.

The toddler's condition can be treated with stem cells from a compatible donor but because of the lack of diversity in the global donor registry, finding a match has been incredibly difficult.

Nguyen said she was moved by the amount of people who came to get swabbed and show their support on Saturday.

"I'm really grateful. These are complete strangers coming to help Minh," she said.

Nguyen said her daughter has shown incredible resilience throughout her time in hospital.

"She's laughing, she's reading, she's playing as much as she can," the mother said.

Friday evening, Nguyen said she started crying at the thought "that she might not outlive me, in terms of lifespan" and Minh asked her why she was sad.

"I said, 'No, I'm just having a bit of allergies,' and she said, 'Please, don't say that, mom, I know you're crying.' I said, 'I just want to stay with you as long as I can.' And she said, 'I'm here for you mom, I'm not leaving you,'" Nguyen recounted.

"She is incredible."

Samuel Sassine, a University of Montreal medical school student who attended Saturday's event, works for a foundation called Swab the World, which was founded by Mai Duong, a woman of Vietnamese descent who also struggled to find a stem cell donor, to encourage people from diverse ethnic backgrounds to become donors.

Sassine said there is currently no one in the world registered with the same DNA background as Minh.

"White people have more chances of living through this disease than other people from other ethnic backgrounds and thatis unacceptable," he said.

"In 2022, ethnic non-equity should not exist, and to live or to not live based on your skin colour should not exist either."

Alex Fong, wholike Surprenant, came to get swabbed on Saturday to see if he could be a potential donor, said Minh's story had move him.

"When I found out that not a lot of Asian donors were doing stem cells, I thought it would be a good idea to come and just participate," said Fong.

Sassinesays he hopes to see more awareness spread in schools about theof the lack of diversity in stem cell donor registries.

He encourageseveryone who is eligible, regardless of ethnicity, to register as a stem cell donor.

In Quebec, stem cell donor registrations are carried out through Hma-Qubec.It involves filling in a questionnaire online and then swabbing cells in your mouth with a kit sent by mail. Once you send it back, you can be part of the donor bank, Sassine explained.

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Stem cell donors volunteer in hopes of saving Montreal toddler with rare blood disease - CBC.ca

Wilmington, Delaware, United States, Transparency Market Research Inc.: Stem cells are bodys raw materials or cells from which all other cells with specialized functions are generated. These cells can be guided to become specific cells that regenerate and repair diseased or damaged tissues in patients suffering from various diseases.

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This stem cell therapy is called regenerative medicine or regenerative therapy to treat several diseases. Stem cell and regenerative therapy promotes the repair response of diseased, injured, or dysfunctional tissue using stem cells or derivatives of stem cells.

Stem cell therapies can be used to treat patients with Parkinson's disease, amyotrophic lateral sclerosis, diabetes type 1, Alzheimer's disease, heart disease, stroke, burns, cancer, and osteoarthritis

In advanced therapies, stem cells are used to replace damaged cells and serve as a replacement method for the donor's immune system to fight several types of blood-related diseases and cancer

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Key Drivers and Restraints of Global Stem Cell and Regenerative Therapy Market

Unclear Regulatory Guidelines Hampers Global Market

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Cell Therapy Segment to Dominate Global Market

Oncology to be Highly Promising Segment

Hospitals to be Major End-user Segment

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North America to Lead Global Stem Cell and Regenerative Therapy Market

Key Manufacturers Operating in Global Stem Cell and Regenerative Therapy Market

Key manufacturers operating in the global market are:

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Stem Cell and Regenerative Therapy Market: Rise in Prevalence of Different Chronic Diseases across the World to Drive the Market - BioSpace