Sodium butyrate efficiently converts fully reprogrammed induced pluripotent stem cells from mouse partially...

Area of Science:

• Stem Cell Biology
• Epigenetics
• Cellular Reprogramming

Background:

• Partially reprogrammed cells (pre-iPSCs) often fail to reach full pluripotency due to epigenetic barriers.
• Overcoming these epigenetic blocks is crucial for efficient induced pluripotent stem cell (iPSC) generation.
• Spontaneous conversion of pre-iPSCs to iPSCs occurs at low frequencies after withdrawal of reprogramming factors.

Purpose of the Study:

• To investigate methods for improving the spontaneous conversion of pre-iPSCs to fully reprogrammed iPSCs.
• To identify small molecules that can reduce epigenetic barriers and enhance reprogramming efficiency.
• To analyze the molecular changes associated with enhanced spontaneous conversion.

Main Methods:

• Established doxycycline (dox)-inducible lentiviral systems to generate iPSC and pre-iPSC lines.
• Cultured pre-iPSCs under dox treatment and withdrawal conditions.
• Treated pre-iPSCs with small molecules: valproic acid (VPA), sodium butyrate (SB), trichostatin (TSA), and 5-aza-2'-deoxycytidine (5-Aza).
• Quantified spontaneous conversion using stage-specific embryonic antigen-1 (SSEA-1) positive colony counts.
• Assessed pluripotency gene expression (Nanog, sox2) and reprogramming factor expression.

Main Results:

• Spontaneous conversion of pre-iPSCs to SSEA-1 positive colonies was low (0.006-0.016%) without small molecule treatment.
• Sodium butyrate (SB) was the most effective small molecule, enhancing SSEA-1 positive colony formation by 32- to 39-fold.
• SB treatment increased the expression of pluripotency genes (sox2, Nanog) and reprogramming factors.
• SB treatment also upregulated cell cycle-related genes in pre-iPSCs.

Conclusions:

• Epigenetic modification using small molecules can significantly improve the spontaneous conversion of partially reprogrammed cells to iPSCs.
• Sodium butyrate (SB) is a potent enhancer of pre-iPSC to iPSC conversion, likely by modulating epigenetic landscapes and gene expression.
• These findings offer a potential strategy to overcome reprogramming barriers and increase the efficiency of iPSC generation.