Differential chondrogenic differentiation between iPSC derived from healthy and OA cartilage is associated with changes in epigenetic regulation and metabolic transcriptomic signatures
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eLife assessment
This study highlights a significant concept whereby a retained memory of disease during stem cell reprogramming (likely via epigenetic modifications) affects the chondrogenic differentiation potential of osteoarthritis (OA)-iMSCs. The evidence supporting the conclusions is compelling, with rigorous RNAseq analysis of genes and signaling pathways. The relevance of this research is highlighted by the valuable role of iPSCs as a potential cell source for regenerative medicine. The work will be of broad interest to skeletal stem cell biologists working on osteoarthritis and cartilage regeneration.
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Abstract
Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted mesenchymal-like progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. Our molecular tracing data demonstrated that dysregulation of epigenetic and metabolic factors seen in OA chondrocytes relative to healthy chondrocytes persisted following iPSC reprogramming and differentiation toward mesenchymal progenitors. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.
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eLife assessment
This study highlights a significant concept whereby a retained memory of disease during stem cell reprogramming (likely via epigenetic modifications) affects the chondrogenic differentiation potential of osteoarthritis (OA)-iMSCs. The evidence supporting the conclusions is compelling, with rigorous RNAseq analysis of genes and signaling pathways. The relevance of this research is highlighted by the valuable role of iPSCs as a potential cell source for regenerative medicine. The work will be of broad interest to skeletal stem cell biologists working on osteoarthritis and cartilage regeneration.
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Reviewer #1 (Public Review):
The contribution of disease-specific factors to the capacity of iPSCs for chondrogenic differentiation is unknown. A better understanding of the underlying mechanism will facilitate approaches to design more effective therapies and interventions to benefit cartilage regeneration.
The authors adequately characterized the stemness of OA-derived iPSC clones compared with previously generated healthy iPSC (AC-iPSCs) based on accepted molecular markers, progenitor properties, and chondrogenic potential pointing to undifferentiated pluripotent phenotype. Clones from AC and OA-iPSCs were then successfully differentiated into mesenchymal progenitor intermediates and displayed similar phenotype characteristics. Immunophenotypic analyses were also performed and confirmed the expression of typical MSC markers in both …Reviewer #1 (Public Review):
The contribution of disease-specific factors to the capacity of iPSCs for chondrogenic differentiation is unknown. A better understanding of the underlying mechanism will facilitate approaches to design more effective therapies and interventions to benefit cartilage regeneration.
The authors adequately characterized the stemness of OA-derived iPSC clones compared with previously generated healthy iPSC (AC-iPSCs) based on accepted molecular markers, progenitor properties, and chondrogenic potential pointing to undifferentiated pluripotent phenotype. Clones from AC and OA-iPSCs were then successfully differentiated into mesenchymal progenitor intermediates and displayed similar phenotype characteristics. Immunophenotypic analyses were also performed and confirmed the expression of typical MSC markers in both population progenitors and the lack of hematopoietic and endothelial markers. In terms of multipotency, both iMSCs differentiated into OBs, adipocytes, and chondrocytes, although AC-iPSCs displayed enhanced chondrogenic potential compared with OA-iPSCs. This was confirmed in the chondrogenic differentiation assay using the pellet culture method and 3D-micromass culture wherein iPSCs derived from healthy chondrocytes displayed significantly higher chondrogenic potential compared with OA-iPSCs. The authors logically concluded that the reduced ECM generation by OA-iMSCs is likely due to retention (or memory) of OA phenotype of the original cell source.RNA-seq analysis of the transcriptome of both AC and OA-iPSCs revealed significant differences between the two cell clones. Similarly, PC analysis suggested that the two populations are genomically distinct. Enrichment of GO terms and KEGG pathway analysis revealed metabolic pathways, epigenetic regulation, and chromatin organization are mostly enriched in AC-iPSCs. These findings suggested that metabolic and epigenetic pathways in AC cells support enhanced chondrogenic differentiation. It should be also noted that the profile of metabolic and epigenetic gene networks exhibited significant differences not only in the terminally differentiated cells but also in the undifferentiated state, further highlighting their distinct chondrogenic potential.
Altogether, using advanced pan-transcriptomic analyses, the authors convincingly demonstrate that distinct expression signature of epigenetic and metabolic marks was detected in healthy iPSCs different from OA-derived iPSCs.
The Implication of epigenetic pre-disposition in OA-iPSC is critically important for designing appropriate strategies to control chondrogenesis and potential cartilage regenerative approaches.
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Reviewer #2 (Public Review):
The manuscript compares the chondrogenic potential of iPSCs derived from human chondrocytes isolated from healthy and osteoarthritic AC tissue. Both iPSCs derived from healthy and osteoarthritic AC tissue exhibit markers of pluripotency and were able to give rise to mesenchymal progenitors, although they had distinct differences in metabolic and chromatin modifier genes, as found by RNA seq analysis. The impact of these transcriptome signatures was functionally reflected in a lower chondrogenic potential of the MSCs derived from OA iPSCs compared to healthy donor (AC) iPSCs. This was assessed based on the reduced expression of hyaline cartilage markers and the reduced deposition of the glycoprotein-rich ECM matrix upon chondrogenic differentiation of day 21 micromass cultures from OA patients compared to …
Reviewer #2 (Public Review):
The manuscript compares the chondrogenic potential of iPSCs derived from human chondrocytes isolated from healthy and osteoarthritic AC tissue. Both iPSCs derived from healthy and osteoarthritic AC tissue exhibit markers of pluripotency and were able to give rise to mesenchymal progenitors, although they had distinct differences in metabolic and chromatin modifier genes, as found by RNA seq analysis. The impact of these transcriptome signatures was functionally reflected in a lower chondrogenic potential of the MSCs derived from OA iPSCs compared to healthy donor (AC) iPSCs. This was assessed based on the reduced expression of hyaline cartilage markers and the reduced deposition of the glycoprotein-rich ECM matrix upon chondrogenic differentiation of day 21 micromass cultures from OA patients compared to healthy donors. The distinct gene expression profiles of OA chondrocytes were also found to be consistent with publicly available RNA-seq data performed on healthy and OA cartilage tissues further confirming that the newly identified differences in epigenetic and metabolic signatures are imprint from healthy and OA-chondrocytes.
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Reviewer #3 (Public Review):
The current manuscript undoubtedly demonstrates that gene expression associated with healthy or diseased donor cartilage used to derive iPSCs influences the iPSCs potential to differentiate to functional chondrocytes. Using comprehensively designed and described experimental approaches they have shown that even though AC-iPSC and OA-iPSC have similar characteristics in terms of stemness and pluripotency, they vary significantly in terms of their chondrogenic differentiation potential. Further, they showed that AC-iMSC and OA-iMSC which are derived from the AC and OA-iPSCs also show similar phenotypic characteristics but differ significantly in terms of their chondrogenic differentiation. The pan-transcriptional analysis confirmed that the AC and OA-iMSC preserve their epigenetic and metabolism-associated …
Reviewer #3 (Public Review):
The current manuscript undoubtedly demonstrates that gene expression associated with healthy or diseased donor cartilage used to derive iPSCs influences the iPSCs potential to differentiate to functional chondrocytes. Using comprehensively designed and described experimental approaches they have shown that even though AC-iPSC and OA-iPSC have similar characteristics in terms of stemness and pluripotency, they vary significantly in terms of their chondrogenic differentiation potential. Further, they showed that AC-iMSC and OA-iMSC which are derived from the AC and OA-iPSCs also show similar phenotypic characteristics but differ significantly in terms of their chondrogenic differentiation. The pan-transcriptional analysis confirmed that the AC and OA-iMSC preserve their epigenetic and metabolism-associated transcriptional memory from AC or OA donor cells which in turn regulate their differentiation to chondrocytes. In summary, these findings have significant implications for designing new approaches to enhance the differentiation potential of iPSCs to desired cells for regenerative research.
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