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January 21, 2010

New Way to Generate Thirty-six Times More Functional Blood Vessel Cells From Human Stem Cells Discovered


In a significant step toward restoring healthy blood circulation to treat a variety of diseases, a team of scientists at Weill Cornell Medical College has developed a new technique and described a novel mechanism for turning human embryonic and pluripotent stem cells into plentiful, functional endothelial cells, which are critical to the formation of blood vessels. Endothelial cells form the interior "lining" of all blood vessels and are the main component of capillaries, the smallest and most abundant vessels. In the near future, the researchers believe, it will be possible to inject these cells into humans to heal damaged organs and tissues. Scientists is hoping to transfer their methods to the clinic within the next five years.

The new approach allows scientists to generate virtually unlimited quantities of durable endothelial cells — more than 40-fold the quantity possible with previous approaches. Based on insights into the genetic mechanisms that regulate how embryonic stem cells form vascular endothelial cells, the approach may also yield new ways to study genetically inherited vascular diseases. The study appears in the advance online issue of Nature Biotechnology

Nature Biotechnology - Expansion and maintenance of human embryonic stem cell–derived endothelial cells by TGFβ inhibition is Id1 dependent

Previous efforts to differentiate human embryonic stem cells (hESCs) into endothelial cells have not achieved sustained expansion and stability of vascular cells. To define vasculogenic developmental pathways and enhance differentiation, we used an endothelial cell–specific VE-cadherin promoter driving green fluorescent protein (GFP) (hVPr-GFP) to screen for factors that promote vascular commitment. In phase 1 of our method, inhibition of transforming growth factor (TGF)β at day 7 of differentiation increases hVPr-GFP+ cells by tenfold. In phase 2, TGFβ inhibition maintains the proliferation and vascular identity of purified endothelial cells, resulting in a net 36-fold expansion of endothelial cells in homogenous monolayers, which exhibited a transcriptional profile of Id1highVEGFR2highVE-cadherin+ ephrinB2+. Using an Id1-YFP hESC reporter line, we showed that TGFβ inhibition sustains Id1 expression in hESC-derived endothelial cells and that Id1 is required for increased proliferation and preservation of endothelial cell commitment. Our approach provides a serum-free method for differentiation and long-term maintenance of hESC-derived endothelial cells at a scale relevant to clinical application.

8 pages of supplemental information



This technique is the first of its kind with serious potential as a treatment for a diverse array of diseases, especially cardiovascular disease, stroke and vascular complications of diabetes," says Dr. Shahin Rafii, the study's senior author. Dr. Rafii is the Arthur B. Belfer Professor in Genetic Medicine and co-director of the Ansary Stem Cell Institute at Weill Cornell Medical College, and an investigator of the Howard Hughes Medical Institute.

In recent years, enormous hopes have been pinned on stem cells as the source of future cures and treatments. Indeed, human embryonic stem cells have the potential to become any one of the more than 200 types of adult cells. However, the factors and pathways that govern their differentiation to abundant derivatives that could be used to repair organs have remained poorly understood.

A major challenge for Dr. Rafii's lab has been to improve their understanding, and hence control, of the differentiation process (how stem cells convert to various cell types), and then to generate enough vascular endothelial cells — many millions — so they can be used therapeutically.

To meet this challenge, the scientists first screened for molecular factors that come into play when stem cells turn into endothelial cells. Their findings led them to a strategy that significantly boosts the efficiency of producing these cells.

Then, the researchers tracked the differentiation process in real-time using a green fluorescent protein marker developed by Dr. Daylon James, the study's first author and assistant research professor in the Department of Reproductive of Medicine at Weill Cornell Medical College. They found that when they exposed stem cells to a compound that blocks TGF-beta (a growth factor involved in cell specialization) at just the right time during cell culturing, the propagation of endothelial cells dramatically increased.

Even more striking, they found that the cells worked properly when injected into mice. The cells were quickly assimilated into the animals' circulatory systems, and functioned alongside normal vasculature.

To achieve long-lasting clinical benefits, there remain additional hurdles to exploiting endothelial cells generated in vitro. Indeed, a fundamental prerequisite to using vascular cells in regenerative medicine has been the proper assembly in vivo of new blood vessels from stem-cell-derived cells, according to Dr. Sina Rabbany, who is an adjunct professor at Weill Cornell Medical College and professor of bioengineering at Hofstra University. Dr. Rabbany emphasizes that, in addition to manipulating biological factors implicated in endothelial cell differentiation, the impact of blood flow on endothelial cells is critical to engineering durable, vascularized organs. With the plentiful supply of endothelial cells that the new methods provide, Dr. Rabbany's team is working to build biological scaffolds that model the microenvironment of the vasculature, so that the vessels they generate will be functional and long-lasting in patients.

Another major obstacle to clinical use of cultured endothelial cells is the potential of immune rejection when the cells are injected into a patient. To address this risk, one approach would be to create a large, genetically diverse bank of human embryonic stem cells that, on demand, could be converted into endothelial cells that are compatible with specific patients.

"Given the success rate our group has shown in generating human embryonic stem cells from donated normal and diseased embryos, this new approach has broad implications not only for regenerative medicine, but also for the study of genetic diseases of the vasculature," states Dr. Zev Rosenwaks, who is director and physician-in-chief of the Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine as well as the director of the Tri-Institutional Stem Cell Initiative Derivation Unit at Weill Cornell Medical College.

The new endothelial cell culture is currently being validated in ongoing research at Weill Cornell using a number of stem cell "lines," or "families" of stem cells. "Employing a highly sophisticated derivation technology, we have been able to generate 11 normal and diseased human embryonic cell lines from discarded embryos at the Tri-Institute Derivation Unit at Weill Cornell," states Dr. Nikica Zaninovic, an assistant professor at the Department of Reproductive of Medicine who is spearheading the human embryonic stem cell derivation effort. Using the new differentiation methods, several of these new embryonic stem cell lines have been turned into vascular cells.

Testing in humans is the next major step in verifying the ability of this breakthrough cell-based approach to restore blood supply to injured organs. Armed with this new technology and under the umbrella of support from the Ansary Stem Cell Institute and Tri-Institutional Stem Cell Initiative (Tri-SCI), this team of scientists is hoping to transfer their methods to the clinic within the next five years.

The current study sheds light on the generation of human embryonic vasculature in ways that have not previously been feasible due to obstacles associated with the use of human embryonic tissue. As Dr. James explains, "The unbiased screening technique we used is a major technological advance that opens up possibilities for discovery of how human embryonic stem cells morph into the specific mature cells that compose the brain, liver, pancreas, and so on. Our general approach can be applied to additional human tissues and help other stem cell research groups develop and maintain specialized cell types in the larger effort to understand human development — and to heal many different kinds of human diseases and injuries."

A page with links to nine videos
Supplementary Video 1. Detection of vasculogenesis in Real-Time: hVPr-GFP+
cells appear at day 5 and connect to form primitive vascular tubules. Human
VPr-GFP EBs were cultured for 5 days and transferred to a live-cell imaging
chamber (TOKAI HIT™) for time-lapse confocal microscopy. These images
demonstrate the emergence of ECs in vasculogenic EBs. Optical Z-stacks were
scanned at an interval of 6 minutes for approximately 35 hours.

Supplementary Video 2. Real-Time Tracking of hESC-derived ECs: Remodeling
of hVPr-GFP+ vessel-like structures in adhering EBs. Human VPr-GFP EBs
were cultured in suspension for 8 days and then transferred to a Matrigel™-coated dish in a live cell imaging chamber. Beginning at day 8, timelapse confocal microscopy revealed the dynamics of endothelial migration in adhering hVPr-GFP EBs. Optical Z-stacks were scanned at an interval of 12 minutes for approximately 100 hours, and then at 3 static time points at days 12, 14 and 16.

Supplementary Video 3. Establishment of vascular patterning: Human VPr-
GFP+ cells form branching microvascular structures with closed lumens. EBs
transferred at differentiation day 8 to adherent conditions and cultured for an additional 17 days generate hVPr-GFP+ cells that are organized into closed, branching micro-vessels.

Supplementary Video 4. Tubulogenesis of human neo-vessels in vitro: Human
VPr-GFP+ cells reorganize into large vessel-like structures following extended differentiation in vitro. Human EBs transferred at differentiation day 8 to adherent conditions and cultured for an additional 17 days form large, vessel-like hVPr-GFP+ structures with closed lumens.

Supplementary Video 5. Whole-well immunodetection of mitotic hESC-derived
ECs in the absence of TGFβ inhibition. Human VPr-GFP EBs were sequentially
stimulated by cytokines and SB431542. ECs were isolated by FACS at day 14,
seeded at a density of 1500 cells per well of a 24 well dish, and then cultured for an additional 5 days in FGF2 and VEGF-A without SB431542. One well was stained with VE-cadherin and phospho-HistoneH3 and scanned using mosaic tiles. Phospho-HistoneH3 positive cells are shown in red and outlined in white. VEcadherin positive cells are shown in green. Nuclear counterstain is shown in blue.

Supplementary Video 6. Whole-well immunodetection of mitotic hESC-derived
ECs in the presence of TGFβ inhibition. Human VPr-GFP EBs were sequentially
stimulated by cytokines and SB431542. ECs were isolated by FACS at day 14,
seeded at a density of 1500 cells per well of a 24 well dish, and then cultured for an additional 5 days in FGF2 and VEGFA with 10μM SB431542. One well was stained with VE-cadherin and phospho-HistoneH3 and scanned using mosaic tiles. Phospho-HistoneH3 positive cells are shown in red and outlined in white. VEcadherin positive cells are shown in green. Nuclear counterstain is shown in blue. Note more than 98% of the cells are ECs expressing VE-cadherin.

Supplementary Video 7. Whole-well immunodetection of non-endothelial cell
types that emerge from hESC-derived ECs in the absence of TGFβ inhibition.
Human VPr-GFP EBs were sequentially stimulated by cytokines and SB431542.
ECs were isolated by FACS at day 14, seeded at a density of 1500 cells per well of a 24 well dish, and then cultured for an additional 5 days in FGF2 and VEGFA without SB431542. One well was stained with VE-cadherin and α-smooth muscle actin and scanned using mosaic tiles. Smooth muscle actin positive cells are shown in red. VE-cadherin positive cells are shown in green. Nuclear counterstain is shown in blue. Note the profound decrease in the number of VE-cadherin+ ECs.

Supplementary Video 8. Human ESC-derived ECs cultured in the presence of
TGFβ inhibitor form functional vessels in vivo. Human VPr-GFP+ cells were
isolated by FACS at day 14 and expanded in monolayer culture and injected in a MatrigelTM plug into immunodeficient mice followed by excision after 10 days after intravital labeling of functional vasculature by GIB4 and UEA lectins. Human VPr-GFP is shown in green; Griffonia simplificolia IB4 lectin is shown in blue; and Ulex europus agglutinin is shown in red.

Supplementary Video 9. Human ESC-derived ECs cultured in the presence of
TGFβ inhibitor form functional vessels in vivo. Human VPr-GFP+ cells were
isolated by FACS at day 14 and expanded in monolayer culture and injected in a MatrigelTM plug into immunodeficient mice followed by excision after 10 days after intravital labeling of functional vasculature by GIB4 lectin. Human VPr-GFP is shown in green; Griffonia simplificolia IB4 lectin is shown in blue.


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