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Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34(pos) Hematopoietic 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.

SUBMITTER: Moslem M

PROVIDER: S-EPMC4491576 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34(pos) Hematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction.

Moslem Mohsen M Eberle Irina I Weber Iuliia I Henschler Reinhard R Cantz Tobias T

Stem cells international 20150622

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-de ...[more]

PMID: 26185499

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Project description:BackgroundDifferentiation of pluripotent stem cells in vitro provides a powerful means to investigate early developmental fates, including hematopoiesis. In particular, the use of a fully defined medium (FDM) would avoid biases induced by unidentified factors contained in serum, and would also allow key molecular mediators involved in such a process to be identified. Our goal was to induce in vitro, the differentiation of human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) into morphologically and phenotypically mature leukocytes and erythrocytes, in the complete absence of serum and feeder cells.Methodology/principal findingsESC and iPSC were sequentially induced in liquid cultures for 4 days with bone morphogenic protein-4, and for 4 days with FLT3-ligand, stem cell factor, thrombopoietin and vascular endothelium growth factor. Cell differentiation status was investigated by both mRNA expression and FACS expression profiles. Cells were further sorted and assayed for their hematopoietic properties in colony-forming unit (CFU) assays. In liquid cultures, cells progressively down-modulated Oct-4 expression while a sizeable cell fraction expressed CD34 de novo. SCL/Tal1 and Runx1 transcripts were exclusively detected in CD34(+) cells. In clonal assays, both ESC and iPSC-derived cells generated CFU, albeit with a 150-fold lower efficacy than cord blood (CB) CD34(+) cells. ESC-derived CD34(+) cells generated myeloid and fully hemoglobinized erythroid cells whereas CD34(-) cells almost exclusively generated small erythroid colonies. Both ESC and iPSC-derived erythroid cells expressed embryonic and fetal globins but were unable to synthesize adult ?-globin in contrast with CB cells, suggesting that they had differentiated from primitive rather than from definitive hematopoietic progenitors.Conclusions/significanceShort-term, animal protein-free culture conditions are sufficient to sustain the differentiation of human ESC and iPSC into primitive hematopoietic progenitors, which, in turn, produce more mature blood cell types. However, additional factors have yet to be identified to allow their differentiation into definitive erythroid cultures.

Project description:BackgroundMesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.MethodsFlow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.ResultsThe stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation.ConclusionsA novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell-cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.

Dataset Information

Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34(pos) Hematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction.

Publications

Mesenchymal Stem/Stromal Cells Derived from Induced Pluripotent Stem Cells Support CD34(pos) Hematopoietic Stem Cell Propagation and Suppress Inflammatory Reaction.

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