Hematopoietic and endothelial differentiation of human induced pluripotent stem cells - PubMed (original) (raw)

Hematopoietic and endothelial differentiation of human induced pluripotent stem cells

Kyung-Dal Choi et al. Stem Cells. 2009 Mar.

Abstract

Induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity for modeling of human diseases in vitro, as well as for developing novel approaches for regenerative therapy based on immunologically compatible cells. In this study, we employed an OP9 differentiation system to characterize the hematopoietic and endothelial differentiation potential of seven human iPSC lines obtained from human fetal, neonatal, and adult fibroblasts through reprogramming with POU5F1, SOX2, NANOG, and LIN28 and compared it with the differentiation potential of five human embryonic stem cell lines (hESC, H1, H7, H9, H13, and H14). Similar to hESCs, all iPSCs generated CD34(+)CD43(+) hematopoietic progenitors and CD31(+)CD43(-) endothelial cells in coculture with OP9. When cultured in semisolid media in the presence of hematopoietic growth factors, iPSC-derived primitive blood cells formed all types of hematopoietic colonies, including GEMM colony-forming cells. Human induced pluripotent cells (hiPSCs)-derived CD43(+) cells could be separated into the following phenotypically defined subsets of primitive hematopoietic cells: CD43(+)CD235a(+)CD41a(+/-) (erythro-megakaryopoietic), lin(-)CD34(+)CD43(+)CD45(-) (multipotent), and lin(-)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. Although we observed some variations in the efficiency of hematopoietic differentiation between different hiPSCs, the pattern of differentiation was very similar in all seven tested lines obtained through reprogramming of human fetal, neonatal, or adult fibroblasts with three or four genes. Although several issues remain to be resolved before iPSC-derived blood cells can be administered to humans for therapeutic purposes, patient-specific iPSCs can already be used for characterization of mechanisms of blood diseases and for identification of molecules that can correct affected genetic networks.

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Figures

Figure 1

Figure 1

Flow cytometric analysis of CD34+ and CD43+ subsets generated in OP9 coculture from (A) hiPSCs (IPS(SK46)-M4-10) and (B) hESCs (H1). There are striking similarities between the subsets of hematopoietic cells developed from these two types of pluripotent cells. Representative analysis of 5 independent experiments is shown.

Figure 2

Figure 2

CFC potential of hESC and hiPSC lines. (A) CFC potential of total cells obtained at day 8 of OP9 coculture. (B) CFC potential of CD43+ and CD43− MACS sorted cells. Results are mean±SE of 3–5 independent experiments. (C) Morphology (upper row, bars are 100 µm) and Wright-stained cytospins (lower row, bars are 50 µm) of CFCs obtained from (SK46)-M4-10 hiPSCs.

Figure 3

Figure 3

Phenotype and function of hiPSC- and hESC-derived endothelial cells. Isolated CD31+CD43− cells were cultured for 7 days in endothelial conditions. (A) Expression of markers of endothelial cells by flow cytometry. (B) Typical morphology of endothelial cell monolayer formed by CD31+CD43− cells (left panels) and immunofluorescent staining of monolayer for VE-cadherin (right panel); cell nuclei visualized by DAPI staining. (C) Vascular tube formation by expanded endothelial cells. All bars are 100µm. Representative experiment of 3 independent experiments is shown.

Figure 4

Figure 4

Flow cytometric analysis of phenotype of hiPSC- (A) and hESC-derived (B) CD34+CD43+CD45− and CD34+CD43+CD45+ primitive hematopoietic cells. Plots show isotype control (open gray) and specific antibody (open black) histograms. The representative experiment of 3–5 independent experiments is shown.

Figure 5

Figure 5

Kinetic analysis of hematopoietic and endothelial development from pluripotent stem cells in OP9 coculture (days 6–12 of differentiation). (A) Percentages of CD43+ hematopoietic and CD31+CD43− endothelial cells within live human (TRA-1-85+7-AAD−) cells (upper row), CD235a/CD41a+, CD235a/CD41a−CD45− and CD235a/CD41a−CD45+ within gated CD43+ cells (middle row) and CD14− cells within gated CD45+ cells (lower row) are shown. (B) Expression of myeloid lineage markers on hiPSC- and hESC-derived CD45+ cells following differentiation in OP9 cocluture.

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