Somatic cells may be reprogrammed into iPSCs using induced expression of transcriptional factors involved in maintaining pluripotency (7
). Expression of Oct3/4, Sox2, Klf4 and c-Myc in somatic cells, reprograms these cells as evidenced by the relengthening of the telomeres (13
) and the acquisition of pluripotency similar to that of embryonic stem cells (15
). Thus, hiPSCs represent a promising cell source for regenerative medicine. These cells are particularly attractive since they can be used to generate patient specific pluripotent cells that do not face an immunological barrier. However, the therapeutic potential of hiPSCs remains largely untested. Recently, hiPSC-derived neurons, cardiomyocytes, and mesenchymal stem cells have shown therapeutic promise in pre-clinical studies(16
). The present study is the first to assess the potential of hiPSC-derived endothelial cells for ischemic vascular disease. We find that hiPSCs can be differentiated into ECs as indicated by typical cobblestone monolayer morphology; expression of EC surface markers and cytoplasmic factors; and manifestation of characteristic endothelial functions (acetylated LDL uptake and capillary-like network formation in matrigel).
When these cells were delivered to the ischemic limb of the mouse by intramuscular injection, they increased increased capillary density and limb perfusion. In contrast, injection of fibroblasts did not improve limb perfusion by comparison to vehicle. We observed some hiPSC-ECs that appeared to incorporate into the microvasculature and others that were in close proximity. However, the observed increase in capillary density could not be accounted for simply by the incorporation of hiPSC-EC into the existing vasculature. Instead, it is likely that the injected cells had a paracrine effect. Indeed, we find that the hiPSC-ECs secrete angiogenic cytokines and growth factors in the presence of hypoxia, as well or better than primary human endothelial cells.
One concern in using iPSC-derived cells is the possibility of contamination of the therapeutic cells with parental iPSCs, which could lead to teratoma formation. We did not observe any tumor formation in any of our animals receiving hiPSC-ECs. However, longer term studies with greater numbers of mice, and/or more sensitive probes to detect undifferentiated cells, are necessary to provide greater assurance that the differentiation process is complete. Also, further study is needed to resolve concerns regarding the possible adverse effects of abnormal imprinting, copy number variation or other somatic mutations arising from the reprogramming or differentiation process.
Using molecular imaging, we observed a reduction in cell number over time in the ischemic limb. Bioluminescence imaging is a sensitive and accurate method for tracking cells in vivo with as few as 500 cells (9
). We found that the number of transplanted cells began to decrease within 24 hours post-transplantation. In order to improve the efficacy of our hiPSC-EC transplantation, we did a second injection at day 7 post-surgery. The transplanted cells face the serious challenge of an unfriendly environment characterized by reduced oxygen and nutrient supply and the presence of various cytotoxic and inflammatory products in the ischemic limb. In addition, although we are using an immunodeficient mouse model, the human cells may stimulate a mild immune reaction and/or may not be receiving appropriate signaling required for their efficient integration into the mouse vasculature.
Adult stem cells (such as those derived from the bone marrow, or circulating mononuclear cells, or mesenchymal stem cells) have been employed in small clinical trials of patients with myocardial ischemia or peripheral arterial disease (19
). Only a few of these trials have been randomized, with large enough numbers of subjects, followed for sufficient period of time, to draw conclusions. These early data have shown proof of concept that cell therapy can provide some benefit in the setting of ischemic syndromes. However, adult stem cells such as endothelial progenitor cells (EPCs) have limited replicative capacity, and are few in number and dysfunctional, in elderly patients with cardiovascular risk factors (25
). By contrast hESCs have unlimited capacity to replicate, can be differentiated into ECs, and in pre-clinical studies have shown beneficial effects in the setting of myocardial or limb ischemia (28
). Previously we have observed that ESC-derived ECs can home to sites of peripheral ischemia, incorporate into the microvasculature, increase capillary density and improve limb perfusion. However, there is an immunogenic barrier for ESC-derivatives and this can pose a challenge in clinical application.
By contrast, hiPSCs can be used to generate autologous therapies, minimizing or eliminating the need for immune suppression after cell transplantation. However, before hiPSCs can be considered for clinical applications, a number of technical issues need to be addressed. Currently most hiPSCs are generated using integrating viral vectors (7
). However, integration of foreign DNA could inadvertently silence indispensable genes, or generate an oncogenic phenotype. Thus, clinical grade therapeutic cells might be derived using nonintegrating episomal vector systems (35
), cell permeant peptides (36
), RNA (38
) and/or small molecules (39
), or RNA-based approaches (40
). In addition, the efficiency and speed of hiPSC generation should be increased, and the fidelity of reprogramming (as assessed by epigenetic, genomic, proteomic and functional studies) must be assured. In this regard, we and others note heterogeneity between hiPSC clones in differentiation potential. These differences are more exaggerated in hiPSC as compared to hESC clones (41
). The reasons for the reduced tendency to differentiate down some lineages in some hiPSC lines are not fully known, but may include factors such as persistent ‘epigenetic memory’ as recently described (42
). Differentiation to functional lineages must be assured, and the risk reduced of transplanting pluripotential or suboptimally differentiated cells. Finally, it is unclear how iPSC-ECs compare in therapeutic efficacy to other clinically relevant cell types such as bone marrow mononuclear cells, endothelial progenitor cells, and mesenchymal stem cells, which each enhance blood flow recovery and improve angiogenesis in the murine hindlimb ischemia model (44
; Suppl. Table I
In conclusion, this is the first study to demonstrate that functional human ECs may be differentiated from hiPSCs, as evidenced by in vitro characterization and in vivo application in a murine model of peripheral arterial disease. This is another step forward toward developing hiPSC-derived cell therapy for vascular regeneration.