Generating functional cells through enhanced interspecies chimerism with human pluripotent stem cells.

Area of Science:

• Stem Cell Biology
• Developmental Biology
• Genetics

Background:

• Interspecies chimerism using human pluripotent stem cells (hPSCs) has faced significant efficiency challenges in generating functional human cells.
• Previous attempts showed limited differentiation and contribution of hPSCs within chimeric embryos.
• The role of specific growth factors in promoting hPSC integration and differentiation across species barriers was not well understood.

Purpose of the Study:

• To investigate methods for enhancing the efficiency of interspecies chimerism for generating functional human cells.
• To determine the effect of MYCN, a pro-growth factor, on hPSC contribution in teratoma co-formation with mouse PSCs.
• To evaluate the potential of MYCN and BCL2 combination (M/B) in promoting hPSC integration and differentiation in various species' embryos.

Main Methods:

• Co-culture of hPSCs with mouse PSCs (mPSCs) and assessment of teratoma formation.
• Introduction of MYCN and/or BCL2 into hPSCs to enhance their growth and survival.
• Injection of modified hPSCs into pre-implantation embryos of mice, rabbits, and pigs.
• Analysis of post-implantation chimeras for hPSC contribution in embryonic and extra-embryonic tissues.
• Cell sorting of human CD34+ blood progenitor cells from chimeric mouse embryos.
• Colony-forming unit (CFU) assays to assess the differentiation potential of chimera-derived human cells.

Main Results:

• MYCN significantly promoted hPSC contribution in teratoma co-formation with mPSCs.
• The combination of MYCN and BCL2 (M/B) greatly enhanced hPSC integration into pre-implantation embryos of mice, rabbits, and pigs.
• M/B-hPSCs substantially contributed to mouse post-implantation chimeras, including embryonic and extra-embryonic tissues.
• Injection into Flk-1+/- mouse embryos further enhanced chimerism, enabling the isolation of live human CD34+ blood progenitor cells.
• Chimera-derived human CD34+ cells demonstrated typical blood cell differentiation potential in CFU assays.

Conclusions:

• MYCN and BCL2 are key factors in enhancing hPSC integration and contribution in interspecies chimeras.
• This study provides a proof-of-concept for generating functional human cells, specifically blood progenitor cells, through enhanced interspecies chimerism.
• The developed method holds promise for future applications in regenerative medicine and disease modeling.