Skin Cells Reprogrammed into Blood Vessel Cells

Induced pluripotent stem cells result from the conversion of adult cells into embryonic-like stem cells by means of genetic engineering techniques. In a nutshell, four specific genes (Oct4, Sox2, and c-Myc and Klf4, or Lin 28 and Nanog; see Takahashi K, Yamanaka S Cell 126:663–676 and Yu J, et al. Science 318:1917–1920) are introduced into adult cells by means of viral vectors (retroviral or adenoviral, see Maherali N, et al. Cell Stem Cell 1:55–70, Okita K, Ichisaka T, Yamanaka S Nature 448:313–317, and Stadtfeld M, et al., Science 322:945–949), plasmids (see Chang CW, et al. Stem Cells 27:1042–104 and Sommer CA, et al. Stem Cells 28:64–74), purified recombinant proteins (Zhou H, et al. Cell Stem Cell 4:381–384), or modified RNA molecules (see Warren L, et al. Cell Stem Cell 7:618–630; Desponts C, Ding S Methods Mol Biol 636:207–218, and Li W, Ding S Trends Pharmacol Sci 31:36–45).

Even though iPSCs have the ability to differentiate into a whole host of cell types, they are limited by their ability to form tumors and the mutations induced by the reprogramming process. Therefore, scientists have been trying to find a way to skip the embryonic-like state when it comes to making cells for therapeutic purposes. Therefore, one paper describes the production of “partial induced pluripotent stem” (PiPS) cells that do not cause tumors in laboratory animals and can still be differentiated into different cell types.

This report was published in the Proceedings of the National Academy of Sciences USA on August 21, 2012. The scientists involved in this work were from the Cardiovascular Division of King’s College London British Heart Foundation Center, UK.

To make PiPS cells, human skin fibroblasts were reprogrammed by means of a plasmid that drove the expression of Oct4, Sox2, Klf4 and c-Myc for 4 days only. This converted the fibroblasts into PiPS cells but not iPSCs. They also found that these PiPS cells could be differentiated into blood vessel cells (endothelial cells or ECs). In fact, PiPS cells differentiated into ECs very readily. The PiPS cell-derived expressed the major proteins normally found in blood vessels, and they no longer expressed any of the genes associated with pluripotency. The ECs also made blood vessels when grown under the right culture conditions (Matrigel plugs), and when injected into laboratory animals, they also made blood vessels.

The next group of experiments examined the mechanisms by which cells become ECs. There is a protein in cells called SETSIP (stands for “similar to SET translocation protein”) that is known to play some role in inducing cells to become ECs. Workers from the laboratory of Qingbo Xu showed that when PiPS cells are treated with a growth factor called VEGF (vascular endothelial growth factor), SETSIP moves into the nucleus and induces the expression of a protein that is specifically expressed on the surface of ECs (VE-cadherin). In fact, when SETSIP expression was decreased with small molecules, no the PiPs cells were completely unable to make blood vessels.

The PiPS cells could even be induced to form pure cultures of ECs. The group went the next step and implanted their PiPS cell-derived ECs into mice that had blocked blood vessels in their hind limbs. The transplanted ECs made blood vessels in the mice and prevented the hind limbs from experiencing damage from a lack of oxygen.  Also, further examination of the implanted cells showed that they indeed did form blood vessels that looked and functioned like normal blood vessels and expressed all the genes  of blood vessels.  See the figure below.

PiPS-ECs improved neovascularization and blood flow recovery in a hindlimb ischemic model. PiPS-ECs, fibroblasts, or medium control (no cells) were injected i.m. into adductors of an ischemic model of SCID mice. (A) Representative color laser Doppler images of superficial blood flow (BF) in lower limbs taken 2 wk after ischemia induction. (B) Line graph shows the time course of postischemic foot BF recovery (calculated as the ratio between ischemic foot BF and contralateral foot BF) in mice given medium as control, fibroblasts, and PiPS-ECs. Statistical analysis showed significantly higher foot BF recovery for PiPS-ECs in comparison with both “no cells” control and fibroblasts at weeks 1 and 2 [data are means ± SEM (n = 6)]. Week 1: **P < 0.01, PiPS-EC vs. “no cells” control; **P < 0.01, PiPS-EC vs. fibroblasts. Week 2: **P < 0.01, PiPS-EC vs “no cell” control; *P < 0.05 PiPS-EC vs. fibroblasts. No significant differences were detected when fibroblasts were compared with “no cells” control. (C) Sections of adductors muscles were stained with CD31 antibody, and capillary density was expressed as the capillary number per mm2 [D; data are means ± SEM (n = 3); *P < 0.05]. (Scale bar, 100 μm.) (E) PiPS-ECs displayed an enhanced engraftment ability compared with fibroblasts when stained and quantified with a human-specific CD31 antibody at six randomly selected microscopic fields (at ×100) [F; data are means ± SEM (n = 3); *P < 0.05]. (Scale bar, 50 μm.)
Thus, this British research group has managed to make stem cells from adult cells that can be differentiated into blood vessels, without the risk of causing tumors. In the concluding words of the authors: “PiPS cells can be a useful cell source for regenerating damaged tissue and vascular engineering ex vivo.” We hope to see safety studies and progression of these cells to clinical trials soon.

See Andriana Margariti, et al., Direct reprogramming of fibroblasts into endothelial cells capable of angiogenesis and reendothelialization in tissue-engineered vessels. PNAS August 21, 2012 vol. 109 no. 34 13793-13798.