Abstract
The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-β-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation.
Highlights
The appeal of regenerative medicine lies in the use of stem cells to replace damaged tissues
The reprogramming is achieved by the introduction of a combination of Octamer Binding Transcription Factor 4 (OCT4 called POU5F1), KLF4, SRY-Box transcription factor 2 (SOX2) and cMYC or OCT4 (POU5F1), KLF4, SOX2 and NANOG to dermal fibroblasts [1,2,3]. iPSCs have been generated by the use of small-molecule drugs [4]
The 3D culture enhances the reprogramming of human mesenchymal stem/stromal cells into iPSCs due to upregulation of the pluripotency factors such as OCT4, SOX2, and NANOG, as compared with embryonic stem cells, the expression levels of these genes are relatively low [9, 10]
Summary
The appeal of regenerative medicine lies in the use of stem cells to replace damaged tissues. In 3D conditions, extracellular physical forces provide a crucial set of signals that control cell structure and functions These forces facilitate cell reprogramming and increase the efficiency of trans-differentiation. The 3D culture enhances the reprogramming of human mesenchymal stem/stromal cells (hMSCs) into iPSCs due to upregulation of the pluripotency factors such as OCT4, SOX2, and NANOG, as compared with embryonic stem cells, the expression levels of these genes are relatively low [9, 10]. The efficacy of dermal fibroblasts to differentiate to osteoblasts is low, as compared to MSCs. Transcriptome analysis showed that dermal fibroblasts which were grown in 3D as spheroids express pluripotency genes such as OCT4, SOX2, NANOG, and Lin and resemble somatic cells that are reprogramed into stem cells [16]. We show that human dermal fibroblasts cultured in 3D as spheroids express pluripotency genes, de-differentiate and transdifferentiate to osteoblasts by a program that requires cystathionine-β-synthase (CBS) mediated release of hydrogen sulfide (H2S)
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