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Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway.

ABSTRACT: A major problem with chondrocytes derived in vitro from stem cells is undesired hypertrophic degeneration, to which articular chondrocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. In vitro differentiation of iPSCs into chondrocytes with an AC-phenotype and resistance to hypertrophy has not been demonstrated so far. Here, we present a novel protocol that succeeded in deriving chondrocytes from human iPSCs without using pro-hypertrophic bone-morphogenetic-proteins. IPSC-chondrocytes had a high cartilage formation capacity and deposited two-fold more proteoglycans per cell than adult ACs. Importantly, cartilage engineered from iPSC-chondrocytes had similar marginal expression of hypertrophic markers (COL10A1, PTH1R, IBSP, ALPL mRNAs) like cartilage from ACs. Collagen X was barely detectable in iPSC-cartilage and 30-fold lower than in hypertrophic cartilage derived from mesenchymal stromal cells (MSCs). Moreover, alkaline phosphatase (ALP) activity remained at basal AC-like levels throughout iPSC chondrogenesis, in contrast to a well-known significant upregulation in hypertrophic MSCs. In line, iPSC-cartilage subjected to mineralizing conditions in vitro showed barely any mineralization, while MSC-derived hypertrophic cartilage mineralized strongly. Low expression of Indian hedgehog (IHH) like in ACs but rising BMP7 expression like in MSCs suggested that phenotype stability was linked to the hedgehog rather than the bone morphogenetic protein (BMP) pathway. Taken together, unlimited amounts of AC-like chondrocytes with a high proteoglycan production reminiscent of juvenile chondrocytes and resistance to hypertrophy and mineralization can now be produced from human iPSCs in vitro. This opens new strategies for cartilage regeneration, disease modeling and pharmacological studies.

SUBMITTER: Diederichs S

PROVIDER: S-EPMC6838640 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway.

Diederichs Solvig S Klampfleuthner Felicia A M FAM Moradi Babak B Richter Wiltrud W

Frontiers in cell and developmental biology 20191101

A major problem with chondrocytes derived <i>in vitro</i> from stem cells is undesired hypertrophic degeneration, to which articular chondrocytes (ACs) are resistant. As progenitors of all adult tissues, induced pluripotent stem cells (iPSCs) are in theory able to form stable articular cartilage. <i>In vitro</i> differentiation of iPSCs into chondrocytes with an AC-phenotype and resistance to hypertrophy has not been demonstrated so far. Here, we present a novel protocol that succeeded in derivi ...[more]

PMID: 31737632

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Project description:BACKGROUND:Intervertebral disc (IVD) degeneration is characterized by an early decrease in cellularity of the nucleus pulposus (NP) region, and associated extracellular matrix changes, reduced hydration, and progressive degeneration. Cell-based IVD therapy has emerged as an area of great interest, with studies reporting regenerative potential for many cell sources, including autologous or allogeneic chondrocytes, primary IVD cells, and stem cells. Few approaches, however, have clear strategies to promote the NP phenotype, in part due to a limited knowledge of the defined markers and differentiation protocols for this lineage. Here, we developed a new protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into NP-like cells in vitro. This differentiation strategy derives from our knowledge of the embryonic notochordal lineage of NP cells as well as strategies used to support healthy NP cell phenotypes for primary cells in vitro. METHODS:An NP-genic phenotype of hiPSCs was promoted in undifferentiated hiPSCs using a stepwise, directed differentiation toward mesodermal, and subsequently notochordal, lineages via chemically defined medium and growth factor supplementation. Fluorescent cell imaging was used to test for pluripotency markers in undifferentiated cells. RT-PCR was used to test for potential cell lineages at the early stage of differentiation. Cells were checked for NP differentiation using immunohistochemistry and histological staining at the end of differentiation. To enrich notochordal progenitor cells, hiPSCs were transduced using lentivirus containing reporter constructs for transcription factor brachyury (T) promoter and green fluorescent protein (GFP) fluorescence, and then sorted on T expression based on GFP intensity by flow cytometry. RESULTS:Periods of pellet culture following initial induction were shown to promote the vacuolated NP cell morphology and NP surface marker expression, including CD24, LM?5, and Basp1. Enrichment of brachyury (T) positive cells using fluorescence-activated cell sorting was shown to further enhance the differentiation efficiency of NP-like cells. CONCLUSIONS:The ability to efficiently differentiate human iPSCs toward NP-like cells may provide insights into the processes of NP cell differentiation and provide a cell source for the development of new therapies for IVD diseases.

Project description:BACKGROUND:The efficiency and quality of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are crucial for regenerative medicine, disease modeling, drug screening, and the study of the development events during cardiac specification. However, their applications have been hampered by the differentiation efficiency, poor maturation, and high interline variability. Recent studies have reported that histamine plays important roles in hematopoietic stem cell proliferation and neutrophil maturation. However, its roles in cardiovascular tissue regeneration have not been thoroughly investigated. In the current study, we identified a novel physiological function of the histamine/histamine 1 receptor (H1R) signal in regulating the differentiation of hiPSC-CMs and heart development. METHODS:Transgenic zebrafish model (cmlc2: mCherry) was treated with histamine and histamine receptor (HR) antagonists. Histological morphology and ultrastructure of zebrafish heart were measured. Histamine-deficient pregnant mice (HDC-/-) were treated with H1R antagonist (pyrilamine) by intragastric administration from E8.5 to E18.5. Cardiac histological morphology and ultrastructure were analyzed in neonatal mice, and cardiac function in adult mice was measured. In vitro, histamine and HR antagonists were administrated in the culture medium during hiPSC-CM differentiation at different stages. The efficiency and maturation of cardiac differentiation were evaluated. Finally, histamine-treated hiPSC-CMs were transplanted into ischemic myocardium to detect the possible therapeutic effect. RESULTS:Administration of H1R antagonist during heart development induced cardiac dysplasia in zebrafish. Furthermore, using histidine decarboxylase (HDC) knockout mice, we examined abnormal swelling of myocardial mitochondria and autophagy formation under the condition of endogenous histamine deficiency. Histamine significantly promoted myocardial differentiation from human induced pluripotent stem cells (hiPSCs) with better structure and function via a H1R-dependent signal. The activation of histamine/H1R signaling pathway augmented hiPSC-derived cardiomyocyte (hiPSC-CM) differentiation through the ERK1/2-STAT3 signaling pathway. In addition, histamine-pre-treated hiPSC-CMs were transplanted into the ischemic hearts of myocardial injured mice and exhibited better survival and myocardial protection. CONCLUSIONS:Thus, these findings indicated that histamine/H1R and its downstream signals were not only involved in cardiac differentiation but also provided a better survival environment for stem cell transplanted into ischemic myocardium.

Dataset Information

Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway.

Publications

Chondral Differentiation of Induced Pluripotent Stem Cells Without Progression Into the Endochondral Pathway.

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