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Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) are transcriptional coactivators in the Hippo signaling pathway. Both are well-known regulators of cell proliferation and organ size control, and they have significant roles in promoting cell proliferation and differentiation. The roles of YAP and TAZ in stem cell pluripotency and differentiation have been extensively studied. However, the upstream mediators of YAP and TAZ are not well understood. Recently, a novel role of YAP in mechanosensing and mechanotransduction has been reported. The present review updates information on the regulation of YAP by mechanical cues such as extracellular matrix stiffness, fluid shear stress, and actin cytoskeleton tension in stem cell behaviors and differentiation. The review explores mesenchymal stem cell fate decisions, pluripotent stem cells (PSCs), self-renewal, pluripotency, and differentiation to blood products. Understanding how cells sense their microenvironment or niche and mimic those microenvironments in vitro could improve the efficiency of producing stem cell products and the efficacy of the products.

Background Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) are two key transcription co-activators of the Hippo pathway. Both were originally characterized as organ size and cell proliferation regulators. Later studies demonstrated that the Hippo pathway may play a role in Drosophila and mammal hematopoiesis. However, the role of the Hippo pathway in human erythropoiesis has not yet been fully elucidated. Methods The role of YAP and TAZ was studied in human erythropoiesis and hematopoietic stem cell (HSC) lineage determination by using mobilized peripheral blood (PB) and cord blood (CB)-derived HSC as a model. HSCs were isolated and cultured in an erythroid differentiation medium for erythroid differentiation and culture in methylcellulose assay for HSC lineage determination study. Results YAP and TAZ were barely detectable in human HSCs, but became highly expressed in pro-erythroblasts and erythroblasts. Depletion or knockdown of YAP and/or TAZ did not affect the ability of HSC lineage specification to erythroid lineage in either methylcellulose assay or liquid culture. However, depletion of YAP and TAZ did impair erythroblast terminal differentiation to erythrocytes and their enucleation. Moreover, ectopic expression of YAP and TAZ in pro-erythroblasts did not exert an apparent effect on erythroid differentiation, expansion, or morphology. Conclusions This study demonstrated that YAP/TAZ plays important role in erythroid maturation and enucleation but is dispensable for lineage determination of human HSCs.

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. In both models at one month of age, Müller glia and microglia mislocalization at dysplastic lesions was similar to that in B6.Cg-Crb1rd8/Pjn mice, while photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg- Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms.

Mutations in the apicobasal polarity gene CRB1 lead to diverse retinal diseases, such as Leber congenital amaurosis, cone-rod dystrophy, retinitis pigmentosa (with and without Coats-like vasculopathy), foveal retinoschisis, macular dystrophy, and pigmented paravenous chorioretinal atrophy. Limited correlation between disease phenotypes and CRB1 alleles, and evidence that patients sharing the same alleles often present with different disease features, suggest that genetic modifiers contribute to clinical variation. Similarly, the retinal phenotype of mice bearing the Crb1 retinal degeneration 8 (rd8) allele varies with genetic background. Here, we initiated a sensitized chemical mutagenesis screen in B6.Cg-Crb1rd8/Pjn, a strain with a mild clinical presentation, to identify genetic modifiers that cause a more severe disease phenotype. Two models from this screen, Tvrm266 and Tvrm323, exhibited increased retinal dysplasia. Genetic mapping with high-throughput exome and candidate-gene sequencing identified causative mutations in Arhgef12 and Prkci, respectively. Epistasis analysis of both strains indicated that the increased dysplastic phenotype required homozygosity of the Crb1rd8 allele. Retinal dysplastic lesions in Tvrm266 mice were smaller and caused less photoreceptor degeneration than those in Tvrm323 mice, which developed an early, large diffuse lesion phenotype. At one month of age, Müller glia and microglia mislocalization at dysplastic lesions in both modifier strains was similar to that in B6.Cg-Crb1rd8/Pjn mice but photoreceptor cell mislocalization was more extensive. External limiting membrane disruption was comparable in Tvrm266 and B6.Cg-Crb1rd8/Pjn mice but milder in Tvrm323 mice. Immunohistological analysis of mice at postnatal day 0 indicated a normal distribution of mitotic cells in Tvrm266 and Tvrm323 mice, suggesting normal early development. Aberrant electroretinography responses were observed in both models but functional decline was significant only in Tvrm323 mice. These results identify Arhgef12 and Prkci as modifier genes that differentially shape Crb1-associated retinal disease, which may be relevant to understanding clinical variability and underlying disease mechanisms in humans.

Background Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into several cell types, including cartilage, fat, and bone. As a common progenitor, MSC differentiation has to be tightly regulated to maintain the balance of their differentiation commitment. It has been reported that the decision process of MSCs into fat and bone cells is competing and reciprocal. Several factors have been suggested as critical factors that affect adipo-osteogenic decision, including melatonin and smad4. Yes-associated protein (YAP) is an important effector protein in the Hippo signaling pathway that acts as a transcriptional regulator by activating the transcription of the genes involved in cell proliferation and anti-apoptosis. The non-canonical role of YAP in regulating bone homeostasis by promoting osteogenesis and suppressing adipogenesis was recently demonstrated in a mouse model. However, it is unclear whether YAP is also crucial for modulating human MSC differentiation to fat and bone. Methods The expression level of YAP during MSC differentiation was modulated using pharmaceutical molecule and genetic experiments through gain- and loss-of-function approaches. Results We demonstrated for the first time that YAP has a non-canonical role in regulating the balance of adipo-osteogenic differentiation of human MSCs. The result from synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy showed unique metabolic fingerprints generated from YAP-targeted differentiated cells that were clearly distinguished from non-manipulated control. Conclusions These results, thus, identify YAP as an important effector protein that regulates human MSC differentiation to fat and bone and suggests the use of FTIR microspectroscopy as a promising technique in stem cell research.

Additional file 3: Figure S3. FTIR spectral signatures of MSCs treated with LPA or DH during differentiation towards osteoblasts or adipocytes. Second derivative spectral (A, E), two-dimensional PCA score plot of all recorded FTIR spectra of DH, LPA, and control cells (B, F). Score loading of PC1 (C, G) and PC2 (D, H) to identify the variable corresponding to wavelength number. Blue dots represent non-treated control, green triangles represent DH, and red squares represent LPA treated cells. Eclipses depicted in the plot define the confidence level with which 95% of the data are allocated. (PPTX 450 kb)

Additional file 1: Figure S1. UC-hMSCs were subjected to immunophenotypic profiling using flow-cytometry. Cells from all three cell lines (A-C) did not express CD34 or CD45, but they were positive for CD73, CD90, and CD105 expression. (PPTX 102 kb)

Yes-associated protein (YAP) is an important transcriptional coactivator in the Hippo signaling pathway. Using CRISPR/Cas9 technology, we established a stable YAP-knockdown (YAP-KD) induced pluripotent stem cell (iPSC) from the MUSIi012-A cell line. The YAP-KD iPSC MUSIi012-A-2 maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and it maintained the normal karyotype. Keywords: YAP, Hippo signaling pathway, iPSC

Background Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and β-thalassemia (HbE/β-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia. Methods We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression. Results The hemoglobin E mutation of HbE/β-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein. Conclusions Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/β-thalassemia in the future. Electronic supplementary material The online version of this article (10.1186/s13287-018-0779-3) contains supplementary material, which is available to authorized users.

Presenting primer and oligo sequences. (DOCX 20 kb)

WWTR1 or TAZ (WWTR1/TAZ) is a transcriptional coactivator that acts as a downstream regulatory target in the Hippo signaling pathway, which plays a pivotal role in regulating cell proliferation and anti-apoptosis. It has been shown in other cell types that WWTR1/TAZ plays a redundant role to its homolog YAP1. Using CRISPR/Cas9 gene editing, we established the WWTR1/TAZ-KO cell line, which features homozygous deletion of WWTR1 gene from human iPSCs. The established WWTR1/YAZ-KO cell line maintained the pluripotent phenotype, the ability to differentiate into all three embryonic germ layers, and normal karyotype.