WH-4-023 (GMP)

Totipotent stem cells are all the cells from the fertilized egg to the 32-cell stage of oogenesis, with unlimited potential for differentiation into cells of various tissues and organs and formation of complete individual.

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Human iPSC reprogramming converts somatic cells into pluripotent cells by introducing defined transcription factors; classic human studies used OCT3/4, SOX2, KLF4, and c-MYC, or OCT4, SOX2, NANOG, and LIN28, and judged reprogramming by embryonic-stem-cell-like morphology, pluripotency-marker expression, normal karyotype, and differentiation into derivatives of the three germ layers. This protocol is framed around non-integrating reprogramming culture, with Sendai virus, episomal plasmids, or synthetic modified mRNA as literature-supported delivery options; Sendai virus is an RNA vector reported to avoid host-genome integration, episomal vectors can generate integration-free human iPSCs, and modified mRNA can reprogram human cells while avoiding genomic modification.

This protocol maintains and expands human pluripotent stem cells under feeder-free, chemically defined conditions using E8 medium and vitronectin-coated culture surfaces; the readout is sustained adherent colony growth with undifferentiated morphology and retained pluripotency-marker expression during serial passaging. E8-based hPSC culture relies on defined soluble factors and matrix-dependent adhesion rather than feeder cells; vitronectin supports hPSC attachment through integrin-mediated interactions, and EDTA passaging dissociates colonies as small aggregates without enzymatic digestion, centrifugation, or routine ROCK-inhibitor treatment.

CRISPR-Cas9 editing of human pluripotent stem cells uses a guide RNA to direct Cas9 to a genomic target, where Cas9 creates a double-strand break that is repaired mainly by non-homologous end joining for knockout mutations or by homology-directed repair when a donor template is supplied for precise knock-in or sequence correction. The readout is generated by genotyping edited bulk populations or single-cell-derived clones, using PCR, sequencing, restriction-based assays, reporter fluorescence, or allele-specific analysis to distinguish unedited alleles, indels, precise donor-mediated edits, biallelic deletions, and unwanted on-target lesions.