Repairing Damaged Organs with New Blood Vessel-Making Stem Cells


A damaged organ usually needs to be removed (spleen or single kidney) or a new organ must be transplanted to replace the damaged organ (liver, heart, lungs, kidney). Wouldn’t it be terrific to inject blood vessel-making stem cells and let the organ heal itself? Such a strategy would render organ transplantation obsolete.

Studies by scientists at the Weill Cornell Medical College in New York have shown that endothelial cells – the cells that line the inside of blood vessels – can drive the regeneration of organ by releasing beneficial, organ-specific molecules. These organ-specific molecules were identified in a genome-wide screen that uncovered all the genes actively expressed in endothelial cells. Many of these genes found in this screen were previously not known to be expressed in endothelial cells. Researchers also found that organ dictate the structure and function of their own blood vessels and this includes the organ-specific repair molecules they elicit from endothelial cells.

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Shahin Rafii, principal investigator of this work, is a professor of genetic medicine and co-director of the medical college’s Ansary Stem Cell Institute and the Tri-SCI Stem Center. Rafii is also a Howard Hughes Medical Institute investigator.

According to Rafii, when an organ in injured, its blood vessels may not have the ability to repair the organ on their own because of the damage to the blood vessels themselves, and the inflammation these same blood vessels might be experiencing.

“Our work suggests that an infusion of engineered endothelial cells could engraft into the injured tissue and acquire the capacity to repair the organ. These studies – along with the first molecule atlas of organ-specific blood vessel cells reported in the Developmental Cell paper (Developmental Cell 26, 204–219, July 29, 2013) – will open up a whole new chapter in translational vascular medicine and will have a major therapeutic application.”

Rafii continued: “Scientists had thought blood vessels in each organ are the same, that they exist to deliver oxygen and nutrients. But they are very different.” According to Rafii, different organs are endowed with blood vessels with unique shape and function and delegated with the difficult task of complying with the metabolic demands of that organ.

In one study from the Rafii lab, nine different tissues were examined, in addition to bone marrow and liver that had undergone a traumatic injury. To examine the blood vessels from each of these tissues, Rafii’s laboratory development a very efficient way to make endothelial cells from embryonic stem cells. Daniel Nolan, the lead author of this work, said that this protocol produced a “a pure population of endothelial cells in a very rapid time frame.”

ECs Derived from hESCs Phenocopy Adult Mouse Tissue-Specific Capillaries (A) Schema of in vitro conditions to support the differentiation and identification of hESC-derived vasculature. hESCs are grown on an E4-ORF1 EC feeder layer and transduced with a VE-Cadherin-Orange reporter gene. VE-Cadherin-Orange+ vascular networks are readily identifiable by day 10. (B) Flow cytometry data depicting the expression of VPR-Orange on hESC-derived CD31+ ECs. These VPR+ ECs have distinct populations based on the expression of either CXCR4 (teal) or CD133 (purple). (C) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs are capable of forming distinct clusters of ECs in hESC cultures. (D) Heat maps of the genes, which were common in their statistically significant differential expression (Benjamini-Hochberg adjusted p < 0.05) between hESC-derived vasculature and adult mouse heart and brain tissues. (E) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs were analyzed for cKit and CD36 levels via flow cytometry. Validation of the higher expression of CD36 and Kit in the CXCR4+ ECs is shown. (F) Heat map of K-Mean clusters depicting the results of de novo motif discovery among non-ECs, CXCR4+VPR+ ECs, and CD133+VPR+ ECs. Candidate binding partners to the motifs are listed.
ECs Derived from hESCs Phenocopy Adult Mouse Tissue-Specific Capillaries.  (A) Schema of in vitro conditions to support the differentiation and identification of hESC-derived vasculature. hESCs are grown on an E4-ORF1 EC feeder layer and transduced with a VE-Cadherin-Orange reporter gene. VE-Cadherin-Orange+ vascular networks are readily identifiable by day 10.  (B) Flow cytometry data depicting the expression of VPR-Orange on hESC-derived CD31+ ECs. These VPR+ ECs have distinct populations based on the expression of either CXCR4 (teal) or CD133 (purple).  (C) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs are capable of forming distinct clusters of ECs in hESC cultures.  (D) Heat maps of the genes, which were common in their statistically significant differential expression (Benjamini-Hochberg adjusted p < 0.05) between hESC-derived vasculature and adult mouse heart and brain tissues.  (E) VPR+CXCR4+CD133− and VPR+CD133+CXCR4− ECs were analyzed for cKit and CD36 levels via flow cytometry. Validation of the higher expression of CD36 and Kit in the CXCR4+ ECs is shown.  (F) Heat map of K-Mean clusters depicting the results of de novo motif discovery among non-ECs, CXCR4+VPR+ ECs, and CD133+VPR+ ECs. Candidate binding partners to the motifs are listed.

From these laboratory-made endothelial cells (ECs), Rafii and his colleagues were able to take snapshots of all the genes expressed in various populations of ECs can compose the different vascular beds of the body. From these studies, Raffi and others discovered that ECs possess specific genes that code for unique growth factors, adhesion molecules, and factors regulating metabolism.

“We knew that these gene products were critical to the health of a particular tissue, but before our study it was not appreciated that these factors originate in the endothelial cells,” said Nolan.

Olivier Elemento, who performed much of the complex computational studies in this paper, noted, “We also found that the healing, or regeneration of tissue, in the liver and in the bone marrow were unexpectedly different – including the repair molecules, known as angiocrine growth factors, that were expressed by the endothelial cells.”

Blood vessels differ among the various organs because the ECs have to constantly adapt to the metabolic, biomechanical, inflammatory, and immunological needs of that particular organ, said Michael Ginsberg, a senior postdoctoral research associate in Rafii’s lab. “And we have now found how endothelial cells have learned to behave differently in each organ and to adjust to the needs of those organs,” he said.

This work from Raffii’s laboratory raises the question as to how ECs have the capacity to adapt to the biological demands of each organ. Is it possible to design “immature” ECs that could allow scientists to identify the means by which particular microenvironmental cues educate these cells to become more specialized endothelial cells?

To address this question, Rafii and his army of graduate students, postdoctoral researchers, technicians, and visiting scientists made ECs from mouse embryonic stem cells and discovered that these cells were responsive to microenvironmental cues, and were also transplantable and functional.

Sina Rabbany, adjunct associate professor of genetic medicine and bioengineering at Weill Cornell Medical College said that embryonic stem cell derived ECs are “very versatile, so they can be transplanted into different tissues, become educated by the tissue, and acquire the characteristics of the native endothelial cells.” These ECs can also be grown in the lab into large numbers.

“We now know what it takes to keep these cells healthy, stable, and viable for transplantation,” said Rabbany.

When the ECs made by Rabbany were transplanted into the livers of laboratory mice, they integrated into the host tissue and become indistinguishable from the native tissue. Similar results were observed when these laboratory-derived ECs were transplanted into kidneys.

Amnion Cells Reprogrammed to Make Blood Vessel Cells


A research team at the Ansary Stem Cell Institute and Weill Cornell Medical College led by Shabin Rafii has succeeded in reprogramming amniotic stem cells into mature blood vessel cells. These blood vessel cells can be banked and potentially used for human therapeutic treatments.

Rafii’s lab has been interested in endothelial cells (the cells that compose blood vessels) for many years, and they used amniotic stem cells for these experiments. Other researchers have shown that introducing three different transcription factors into pluripotent stem cells (ETV2, FLI1 and ERG1), can drive the stem cells to differentiate into induced vascular endothelial cells (iVECs). These cells, however, were immature and were not completely differentiated versions of endothelial cells. Furthermore, the iVECs were unstable because they tended to differentiate into non-endothelial cell types while in culture. Therefore, Rafii was sure this regiment was on the right track, but it needed tweaking.

To perfect this protocol, Rafii and co-workers chose to work with amniotic stem cells. Amniotic stem cells are a wonderfully robust stem cell population that have the ability to form a wide variety of cell types and do not form tumors. During development, the embryo is surrounded by a thin membrane called the amniotic membrane. At Carnegie Stage 6, at the end of the 2nd week of development (day 13-14), the embryo is about 0.2 millimeters long. A veil of tissue grows over the disc-like structure at the very top of the embryo. This veil of tissue is called the amnion and the cavity is generates is the amniotic cavity. The embryo grows within this cavity, suspended in amniotic fluid, and as the embryo grows, the amnion grows with it, as does the size of the amniotic cavity within which he embryo remains suspended all the way through embryonic and fetal development until birth.

First of all, Rafii and colleagues transiently expressed ETV2 in the amniotic stem cells and found that they differentiated into iVECs. Therefore, they tried co-expressing FLI1/ERG1 with ETV2, and found that the cells expressed several vascular-specific proteins and assumed a shape that matched mature endothelial cells (ECs). Next, they briefly shut off TGFβ signaling. This pushed the cells over the edge and they became endothelial cells. Their success rate was around 20 percent, which is astounding, since most reprogramming protocols usually sport as success rate of around 1 percent.

When Rafii and his colleagues examined the gene expression profile of the endothelial cells derived from amniotic stem cells, they found that their cultured endothelial cells were very similar to adult endothelial cells: vascular-specific genes were expressed and nonvascular genes were silenced.

Functional assays further confirmed that they are converted amniotic stem cells into endothelial cells. When Raffi and others gave these cells a gel-like matrix called Matrigel, they formed a filigree of blood vessels in the Matrigel plug. When they transplanted their cultured endothelial cells in a living animal, they were able to regenerate the internal sinuses and vasculature of a sick liver. Thus this protocol reprogrammed mature amniotic stem cells into fully functional endothelial cells clinical-scale expansion potential.

The therapeutic potential of this work is certainly not lost on Rafii. He explained, “There is no curative treatment available for patients with vascular diseases, and the common denominator to all these disorders is dysfunction of blood vessels, specifically endothelial cells that are the building blocks of the vessels.”

These cultured endothelial cells, however, do more than just make blood vessels. They produce growth factors such as vascular endothelial growth factor (VEGF) that promote the maintenance, repair, and regeneration of the vasculature. Damaged blood vessels may not be able to stimulate the repair of the organs they service, but newly infused endothelial cells could.

Also tissue engineers tend to grow tissues and artificial organs in porous three-dimensional scaffolds. These cultured endothelial cells could easily form functional blood vessels on such surfaces if they were introduced into an injured organ. Rafii noted that his cultured endothelial cells could be a huge boost for translational vascular medicine. He optimistically predicts that four years of preclinical work could persuade the FDA to approve human clinical trials in which his cells are used to treat vascular disorders. Also banking tissue-typed amnion-derived vascular endothelial cells could establish an inventory of cells for the treatment of diverse disorders.

This work also suggests that amniotic stem cells could potentially be reprogrammed to efficiently form other cell types as well.