ROCK inhibition facilitates the generation of human-induced pluripotent stem cells in a defined, feeder-, and serum-free system

Abstract

Human-induced pluripotent stem cells (iPSCs) generated from human adult somatic cells through reprogramming hold great promises for future regenerative medicine. However, exposure of human iPSCs to animal feeder and serum in the process of their generation and maintenance imposes risk of transmitting animal pathogens to human subjects, thus hindering the potential therapeutic applications. Here, we report the successful generation of human iPSCs in a feeder-independent culture system with defined factors. Two stable human iPSC lines were established from primary human dermal fibroblasts of two healthy volunteers. These human iPSCs expressed a panel of pluripotency markers including stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase, while maintaining normal karyotypes and the exogenous reprogramming factors being silenced. In addition, these human iPSCs can differentiate along lineages representative of the three embryonic germ layers upon formation of embryoid bodies, indicating their pluripotency. Furthermore, subcutaneous transplantation of these cells into immunodeficient mice resulted in teratoma formation in 6 to 8 weeks. Our findings are an important step toward generating patient-specific iPSCs in a more clinically compliant manner by eliminating the need of animal feeder cells and animal serum.

FIG. 1.

Initial trials of human iPSC generation in feeder-independent, serum-free condition. (A–F) Immunofluorescence analysis showed successful lentivirus transduction of reprogramming transcription factors in primary human dermal fibroblasts. (G–H) Typical clusters of cells resembling human ESC colonies appeared 10 days posttransduction in lower and higher magnification (circle enclosed). (I) Rounding up and detachment of putative iPSC clusters in the absence of Y-27632 at day 10 (arrowheads).

FIG. 2.

Generation of human iPSCs with feeder-independent, serum-free condition with defined medium. (A) Outline of experimental design. Freshly isolated human dermal fibroblasts in mTeSR™1 medium supplemented with 50 ng/mL bFGF were transduced with lentiviruses. Seven days posttransduction, ROCK inhibitor Y-27632 was applied. (B) Colonies of human iPSCs (arrowheads) in the background of incomplete reprogrammed cells 28 days posttransduction. (C) Hand-picked putative human iPSC colony subcultured to new Matrigel™-coated wells. (D–F) Morphology of hand-picked human iPSC colony 5 days (D), 10 days (E) after subculture, and at 10 passages. (G) Intensive alkaline phosphatase expression in human iPSC colony (purple color).

FIG. 3.

The number of ESC-like clusters 14 days and 28 days after reprogramming of primary human dermal fibroblasts obtained from two healthy Chinese males with and without ROCK inhibition (n = 3).

FIG. 4.

Characterization of the human iPSC lines. Immunofluorescence analysis of human iPSC lines (A) KS1, (B) KS2. (C) H9 hESC as a comparative control. The colonies expressed embryonic stem cell markers including Oct4, Nanog, KLF, SSEA-4, TRA-1-60, and SOX2. (D) Flow cytometry analysis of patients-specific human iPSC lines for pluripotency markers: Oct-4, SSEA-4, TRA-1-60, and TRA-1-81.

FIG. 4.

Characterization of the human iPSC lines. Immunofluorescence analysis of human iPSC lines (A) KS1, (B) KS2. (C) H9 hESC as a comparative control. The colonies expressed embryonic stem cell markers including Oct4, Nanog, KLF, SSEA-4, TRA-1-60, and SOX2. (D) Flow cytometry analysis of patients-specific human iPSC lines for pluripotency markers: Oct-4, SSEA-4, TRA-1-60, and TRA-1-81.

FIG. 5.

(A) Cytogenetic analysis of human iPSC lines. Both KS-1 iPSCs and KS-2 iPSCs remained in normal karyotypes. (B) Oct-4 promoter methylation analysis with bisulfate pyro-sequencing in two parent fibroblast lines (KS-1 and KS-2), human iPSC lines (KS-1 iPSC and KS-2 iPSC), endothelial progenitor cells (EPCs) derived from the two human iPSC lines (KS-1 iPSC-EPC and KS-2 iPSC-EPC). H9 hESC served as control. (C) RT-PCR analyses of the endogenous and exogenous level of OCT4 and Nanog of KS-1 iPSC at day 14 after transduction, KS-1 iPSC at passage 20 (P20), and KS-2 iPSC at passage 11 (P11). H9 hESC served as control.

FIG. 6.

Differentiation of human iPSCs generated under feeder-independent, serum-free condition with defined medium. (A) RT-PCR analyses of undifferentiated human iPSC lines and the embryoid bodies for REX1 (pluripotency marker), as well as the markers for the three embryonic germ layers [Nestin, ectoderm; Intercellular adhesion molecule 1 (ICAM1), mesoderm; Indian hedgehog homolog (IHH) and alpha-fetoprotein (AFP), endoderm]. GAPDH served as internal control and NC is a negative control for the PCR. (B) In vitro differentiation: embryoid body formation in suspension culture with spontaneously differentiation into the three germ layers: smooth muscle actin (mesoderm), nestin (ectoderm), and alpha-fetoprotein (endoderm). In addition, human iPSC spontaneously differentiated into cardiomyocytes (troponin) and endothelial cells (vWF) (arrowheads for positive stained cells). (C) Human iPSC-induced teratoma formation 5 weeks after subcutaneous injection in NOD/SCID mice. (D) Immunohistological staining of the three grem layers (arrowheads) in the teratoma generated by two different human iPSCs generated under feeder-independent, serum-free condition with defined medium.

FIG. 6.

Differentiation of human iPSCs generated under feeder-independent, serum-free condition with defined medium. (A) RT-PCR analyses of undifferentiated human iPSC lines and the embryoid bodies for REX1 (pluripotency marker), as well as the markers for the three embryonic germ layers [Nestin, ectoderm; Intercellular adhesion molecule 1 (ICAM1), mesoderm; Indian hedgehog homolog (IHH) and alpha-fetoprotein (AFP), endoderm]. GAPDH served as internal control and NC is a negative control for the PCR. (B) In vitro differentiation: embryoid body formation in suspension culture with spontaneously differentiation into the three germ layers: smooth muscle actin (mesoderm), nestin (ectoderm), and alpha-fetoprotein (endoderm). In addition, human iPSC spontaneously differentiated into cardiomyocytes (troponin) and endothelial cells (vWF) (arrowheads for positive stained cells). (C) Human iPSC-induced teratoma formation 5 weeks after subcutaneous injection in NOD/SCID mice. (D) Immunohistological staining of the three grem layers (arrowheads) in the teratoma generated by two different human iPSCs generated under feeder-independent, serum-free condition with defined medium.

FIG. 7.

Differentiation of human iPSCs to endothelial progenitor cells (EPCs). Fluorescence staining analysis to characterize the KS1-iPSC-derived EPCs. (A) Bright-field, (B) lectin, (C) Di-acetyl-LDL, (D) merge of B and C, (E) smooth muscle actin (SMA), and (F) von-Willebrand factor (vWF). (G) Functional assays of endothelial progenitor cells (EPCs) derived from human iPSCs (KS-2-iPSC-EPCs) using tube formation assay and cell migration assay. H1 hESC-derived EPC served as control.