Hydrogen sulfide facilitates reprogramming and trans-differentiation in 3D dermal fibroblast

Area of Science:

• Cell Biology
• Stem Cell Research
• Biochemistry

Background:

• Two-dimensional (2D) cell cultures exhibit limited cell reprogramming efficiency.
• Cellular stemness is influenced by microenvironmental factors, suggesting 3D cultures may improve reprogramming.
• The specific mechanisms by which 3D environments affect fibroblast reprogramming remain unclear.

Purpose of the Study:

• To investigate how 3D culture conditions influence the reprogramming potential of dermal fibroblasts.
• To elucidate the role of specific metabolic pathways and molecules in 3D-mediated cell reprogramming.
• To explore the potential of 3D cultures for cell trans-differentiation.

Main Methods:

• Formation of 3D dermal fibroblast spheroids using ultra-low attachment conditions.
• Measurement of lactate production and hydrogen sulfide (H2S) levels.
• Gene expression analysis of pluripotency markers (OCT4, NANOG, SOX2).
• RNA interference (RNAi) to knockdown cystathionine-β-synthase (CBS).
• Treatment with H2S donors (NaHS, diallyl trisulfide - DATS).

Main Results:

• 3D fibroblast spheroids showed increased lactate production, a prerequisite for reprogramming.
• Upregulation of pluripotency genes (OCT4, NANOG, SOX2) and cystathionine-β-synthase (CBS) with subsequent H2S production was observed.
• CBS knockdown reduced lactic acid and H2S, decreasing OCT4 and NANOG expression.
• H2S donors (NaHS, DATS) enhanced OCT4 expression and supported osteogenic trans-differentiation.

Conclusions:

• Cystathionine-β-synthase (CBS) mediated release of hydrogen sulfide (H2S) is a key regulator of dermal fibroblast reprogramming in 3D cultures.
• 3D culture environments, through metabolic alterations like increased H2S, promote stemness and facilitate cell trans-differentiation.
• This study highlights the potential of 3D cell culture models for advancing stem cell research and regenerative medicine.