Induction and Isolation of Vascular Cells From Human Induced Pluripotent Stem Cells—Brief Report
Objective— Induced pluripotent stem (iPS) cells are a novel stem cell population derived from human adult somatic cells through reprogramming using a defined set of transcription factors. Our aim was to determine the features of the directed differentiation of human iPS cells into vascular endothelial cells (ECs) and mural cells (MCs), and to compare that process with human embryonic stem (hES) cells.
Methods and Results— We previously established a system for differentiating hES cells into vascular cells. We applied this system to human iPS cells and examined their directed differentiation. After differentiation, TRA1–60− Flk1+ cells emerged and divided into VE-cadherin-positive and -negative populations. The former were also positive for CD34, CD31, and eNOS and were consistent with ECs. The latter differentiated into MCs, which expressed smooth muscle α-actin and calponin after further differentiation. The efficiency of the differentiation was comparable to that of human ES cells.
Conclusions— We succeeded in inducing and isolating human vascular cells from iPS cells and indicate that the properties of human iPS cell differentiation into vascular cells are nearly identical to those of hES cells. This work will contribute to our understanding of human vascular differentiation/development and to the development of vascular regenerative medicine.
Pluripotent embryonic stem (ES) cells are thought to represent a potentially unlimited pool from which to derive cells for new treatments in the area of regenerative medicine and for investigation of cell development/differentiation. We previously described the process by which mouse, monkey and human embryonic stem (ES) cells differentiate into vascular cells.1,2,3 In addition, we used the hindlimb ischemia model with immunodeficient mice to demonstrate that transplanted vascular endothelial cells (ECs) and mural cells (MCs) derived from human (h)ES cells could be successfully incorporated into the host vasculature and significantly accelerate improvements in local blood flow.3,4 However, immunologic and ethical problems remain to be overcome before clinical application.
Recently, novel ES cell-like pluripotent cells were generated from mouse skin fibroblasts by introducing 4 transcription factors.5 Termed induced pluripotent stem (iPS) cells, they were subsequently generated from human skin fibroblasts.6,7 At present, the properties of human iPS cell differentiation into vascular cells remain unknown. To address that issue, we investigated the differentiation of human iPS cells into ECs and MCs using our differentiation system previously developed for hES cells.
Materials and Methods
hES, human iPS, and OP9 feeder cells were all established and maintained as described previously.6,8,9 To induce differentiation, hES or iPS cells were cultured on an OP9 feeder layer as described previously.3
Flow Cytometry and Cell Sorting
Flow cytometric analysis and cell sorting were performed as described previously.1,9
Cultured cells were stained with various monoclonal antibodies as described.1,9
For details regarding cell culture, RT-PCR, and the antibodies used in flow cytometry and immunohistochemistry, please see the supplemental material (available online at http://atvb.ahajournals.org).
We investigated 3 hES cell lines (H9, HES3, and KhES1) and 4 iPS cell lines (201B6, 201B7, 253G1, and 253G4).5,6 201B6 (B6) and 201B7 (B7) cells were generated from human skin fibroblasts by transfection with 4 transcription factors (Oct3/4, Sox2, Klf4, c-Myc), whereas 253G1 (G1) and 253G4 (G4) were generated using only 3 factors (c-Myc was omitted).10 The morphology of these 4 lines did not differ from hES cells, and they were also positive for hES cell markers (supplemental Figure I). We induced differentiation of these iPS cell lines in an in vitro 2D culture system previously established for differentiation of hES cells into vascular cells.3 After 10 days of differentiation, cells positive for Flk1 (also designated VEGF receptor-2) and the EC marker VE-cadherin emerged and accounted for 1% to 5% of the cells (Figure 1A through 1G). We noted no differences in the differentiation of the B and G lines, and both were comparable to the hES lines (Figure 1A through 1G). The Flk1+ VE-cadherin+ cell population was also positive for CD31 and CD34 (Figure 1H and 1I), and negative for the ES cell marker tumor rejection antigen 1–60 (TRA1–60). We sorted those cells and recultured with VEGF, and found that they formed a network-like structure on Matrigel, in vitro (Figure 2A), and had a cobblestone appearance when confluent on collagen IV-coated dishes (Figure 2B). Immunofluorescent staining for CD31 produced a characteristic marginal staining pattern (Figure 2C), and staining for endothelial NO synthase produced a cytoplasmic pattern (Figure 2D). Based on these observations, the cells were consistent with ECs. Subsequent RT-PCR analysis of EC markers revealed that both human iPS-derived and hES-derived ECs expressed VE-cadherin, CD31, von Willebrand factor (vWF), and CD34 at levels similar to those seen in adult ECs (supplemental Figure II).
We next sorted for Flk1+ VE-cadherin− TRA1–60− cells on day 10 of differentiation and then induced differentiation into MCs using PDGF-BB as described previously.3,4 Once differentiated, these cells stained positively for αSMA and calponin and were therefore consistent with MCs (Figure 2E and 2F). Both human iPS-derived and hES-derived MCs expressed the vascular smooth cell markers at levels similar to those seen in adult vascular smooth muscle cells (supplemental Figure II).
The establishment of iPS cells opened a new avenue for regenerative medicine and stem cell biology. The directional differentiation of mouse iPS cells into vascular cells was recently reported.11 In the present study, we have shown that human iPS cells can be directionally differentiated into vascular ECs and MCs by applying the same methods we established for hES cells.3 We previously reported that the differentiation kinetics of primate ES cells to vascular cells is not equal to that of mouse ES cells.2,3 To further clarify the differentiation process in human beings and to determine the possible clinical application of iPS cells, investigation of human iPS cells is essential because some characters were significantly different between mouse and human iPS cells as ES cells.
In contrast to human ES cells, iPS cells can be established from every human being irrespective of their genetic backgrounds. The establishment of in vitro differentiation system of human vascular cells from human iPS cells should make it possible to dissect out cellular mechanisms in human vascular development and diseased states such as arteriosclerosis. The establishment of iPS cell lines from patients with inherited diseases presenting vascular abnormality should enable clarification of their pathogenesis. In addition, because they overcome the immunologic and ethical problems associated with human ES cells, our study should also contribute to the development of novel patient-specific cell based vascular regenerative therapies.
Several issues remain to be resolved before human iPS-derived vascular cells can be administered to humans, however. Although we observed no reappearance of undifferentiated or tumor cell-like structures in the in vitro cultures, when we examined the mRNA expression of the transgenes during differentiation experiments, we occasionally observed upregulation of the transgenic mRNA (supplemental Figure III). The safety of iPS cells needs to be confirmed for each iPS cell line, both in vitro and in vivo.
In conclusion, we succeeded in inducing and isolating human vascular cells from iPS cells and indicate that the properties of differentiation are nearly identical to those of hES cells. This work will contribute to our understanding of human vascular differentiation/development and to the development of vascular regenerative medicine.
We described additional discussions about the safety of iPS cells in the supplemental material.
We thank Yoshie Fukuchi for her technical assistance. The antihuman Flk1 antibody (KM1668) was a generous gift from Kyowa Hakko Co Ltd.
Sources of Funding
This work was supported by the project for realization of regenerative medicine of the Ministry of Education, Culture, Sports, Science, and Technology, Japan, Grants-in-Aid for Scientific Research from the Ministry of Health, Labor, and Welfare, the Ministry of Education, Culture, Sports, Science, and Technology, the Takeda Science Foundation, the Japan Cardiovascular Research Foundation, and the Smoking Research Foundation.
Received December 4, 2008; revision accepted April 16, 2009.
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