Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34^posHematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction

Abstract

Mesenchymal stem/stromal cells (MSCs) represent a promising cell source for research and therapeutic applications, but their restricted ex vivo propagation capabilities limit putative applications. Substantial self-renewing of stem cells can be achieved by reprogramming cells into induced pluripotent stem cells (iPSCs) that can be easily expanded as undifferentiated cells even in mass culture. Here, we investigated a differentiation protocol enabling the generation and selection of human iPSC-derived MSCs exhibiting relevant surface marker expression profiles (CD105 and CD73) and functional characteristics. We generated such iPSC-MSCs from fibroblasts and bone marrow MSCs utilizing two different reprogramming constructs. All such iPSC-MSCs exhibited the characteristics of normal bone marrow-derived (BM) MSCs. In direct comparison to BM-MSCs our iPSC-MSCs exhibited a similar surface marker expression profile but shorter doubling times without reaching senescence within 20 passages. Considering functional capabilities, iPSC-MSCs provided supportive feeder layer for CD34+ hematopoietic stem cells' self-renewal and colony forming capacities. Furthermore, iPSC-MSCs gained immunomodulatory function to suppress CD4+ cell proliferation, reduce proinflammatory cytokines in mixed lymphocyte reaction, and increase regulatory CD4+/CD69+/CD25+ T-lymphocyte population. In conclusion, we generated fully functional MSCs from various iPSC lines irrespective of their starting cell source or reprogramming factor composition and we suggest that such iPSC-MSCs allow repetitive cell applications for advanced therapeutic approaches.

Kindlin-2 Modulates the Survival, Differentiation, and Migration of Induced Pluripotent Cell-Derived Mesenchymal Stromal Cells

“…Previously, we showed that iPSC-MSCs displayed a shorter doubling time than BM-MSCs and reached senescence at later passages than BM-MSCs [10]. To investigate the effects of Kindlin-2 on proliferation and survival in iPS-MSCs, we performed a BrdU incorporation assay (Figure 2(a)) that showed a significant increase after Kindlin-2 Flag transfection compared to the vector control.…”

“…Cells were split weekly using collagenase IV (Thermo Fisher), and cells were plated on Matrigel-coated (Corning) plates. Differentiation/enrichment of iPSCs to MSCs was conducted as described [10]. In brief, human iPSC colonies grown on Matrigel were maintained with MSC induction media consisting of DMEM (low-glucose, Sigma-Aldrich, Darmstadt, Germany), 10% defined fetal bovine serum (FBS, Stem Cell Technologies, Vancouver, BC, Canada), 1% nonessential amino acids, 1% penicillin-streptomycin, and 2 ng/mL human recombinant bFGF for 7 days.…”

“…Twenty-four hours after transfection of iPSC-MSCs, mixed lymphocyte reaction (MLR) cultures were inoculated with 5 × 10 4 mitomycin C-treated (Sigma-Aldrich) human peripheral blood mononuclear cells (PBMCs) as stimulators and 2 × 10 5 human CD8 + T-cells isolated from normal blood donors after informed consent in 96-well round-bottom plates in 200  μ L of complete medium containing RPMI 1640 (Life Technologies) supplemented with 0.1 mM β -mercaptoethanol, 10% FBS, GlutaMAX I (Life Technologies), 100 U/mL penicillin, and 100  μ g/mL streptomycin in the presence or absence of iPSC-MSCs and BM-MSCs as previously described [10]. Cultures were incubated for 5 days in 37°C/5% CO 2 .…”

“…In a previous study, we characterized the differentiation of iPSCs towards MSCs to obtain a functional substitute for ex vivo MSCs [7, 10]. We have shown that iPSCs can be differentiated into MSCs, including development from “epithelial-like” iPSCs towards spindle-shaped MSCs that are capable of proliferation in an undifferentiated stage and of induction into multilineage differentiation.…”

“…We have shown that iPSCs can be differentiated into MSCs, including development from “epithelial-like” iPSCs towards spindle-shaped MSCs that are capable of proliferation in an undifferentiated stage and of induction into multilineage differentiation. Moreover, iPSC-MSCs showed similar hematopoietic support and immunomodulatory effects to BM-MSCs [10]. In this study, we aimed to modify Kindlin-2 expression in iPSC-MSCs to modulate their proliferative and functional properties.…”