We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.
Beta‐thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with β‐thalassemia (β‐thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site‐specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence‐specific nuclease to enhance correction of a specific HBB mutation by homology‐directed repair (HDR) is becoming straightforward, targeting various HBB mutations of β‐thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two β‐thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in β‐thal and SCD. stem cells translational medicine 2018;7:87–97
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