Haploid Human Embryonic Stem Cell Derivation via Parthenogenetic Activation
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Solution Overview
Problem
The derivation of haploid human embryonic stem (ES) cell lines has been hindered by the limited availability of human oocytes and the challenges of genomic stability and differentiation potential, limiting the use of CRISPR/Cas9-mediated mutagenesis and gene trap methods in diploid cells for loss-of-function genetic screens.
Innovation Solution
Artificial activation of unfertilized metaphase II human oocytes results in efficient development to the blastocyst stage, allowing the derivation of parthenogenetic ES cell lines with a normal haploid karyotype, which are maintained and enriched through methods like metaphase spread analysis, flow cytometry, and sorting based on cell ploidy to produce enriched and pure populations of haploid human ES cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If artificial activation of unfertilized metaphase II human oocytes is used to derive haploid ES cell lines, then genomic stability and pluripotency are improved, but the limited availability of human oocytes remains a constraint
Solution Approach 1:
The patent performs preliminary sorting of haploid cells from parthenogenetic ES cell populations before establishing permanent cell lines. By using flow cytometry and metaphase spread analysis to identify and isolate haploid cells early in the derivation process, the method ensures genomic stability is established from the outset, preventing later contamination by diploid cells that would compromise the cell line's reliability.
2Productivity
If CRISPR/Cas9-mediated mutagenesis is used in diploid cells, then gene disruption efficiency is improved, but functional gene disruption becomes less efficient due to allele differences
Solution Approach 1:
The patent extracts the redundancy of having two alleles by using haploid ES cells instead of diploid cells. In haploid cells, there is only one copy of each gene, eliminating the need to account for allele differences, heterozygosity, or dominant-negative effects. This simplification makes CRISPR/Cas9 mutagenesis and other genetic manipulation methods significantly more efficient and reliable, as any genetic modification immediately affects the single functional copy of the gene.
3Adaptability or versatility
If haploid ES cell lines are derived, then loss-of-function genetic screens are improved, but cell sorting complexity increases due to the need for metaphase spread analysis and flow cytometry
Solution Approach 1:
The patent performs preliminary enrichment of haploid cells during the ES cell line derivation process itself, rather than requiring complex sorting at every subsequent passage. By establishing haploid-enriched cell lines through initial sorting and maintenance protocols, the method reduces the need for repeated complex sorting operations, thereby simplifying downstream applications while preserving the advantages of haploid genetics for loss-of-function screens.
4Stability of the object's composition
If diploidy-dependent adaptations such as parental imprinting are present, then genome function is stabilized, but development of haploid uniparental embryos is restricted
Solution Approach 1:
The patent uses parthenogenetic activation to create haploid embryos with two identical maternal genomes instead of the normal one maternal and one paternal genome. This 'copying' of the maternal genome bypasses the parental imprinting restrictions that normally prevent haploid development, as the imprinted genes receive identical epigenetic marks from the same parent. This allows haploid ES cells to be derived and maintained despite the presence of diploidy-dependent imprinting mechanisms in the human genome.
Data Source
AI summary
Haploid human embryonic stem cells and cell lines, haploid multipotent human cells, and haploid differentiated human cells are provided. In addition, methods of making and using the haploid human cells are provided.