OGT and OGA gene-edited human induced pluripotent stem cells for dissecting the functional roles of O-GlcNAcylation in hematopoiesis.

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor O-GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of OGA-depleted (OGAi) and OGT-depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34+ hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O-GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O-GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O-GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of O-GlcNAcylation.

An expedition in the jungle of pluripotent stem cells of non-human primates.

For nearly three decades, more than 80 embryonic stem cell lines and more than 100 induced pluripotent stem cell lines have been derived from New World monkeys, Old World monkeys, and great apes. In this comprehensive review, we examine these cell lines originating from marmoset, cynomolgus macaque, rhesus macaque, pig-tailed macaque, Japanese macaque, African green monkey, baboon, chimpanzee, bonobo, gorilla, and orangutan. We outline the methodologies implemented for their establishment, the culture protocols for their long-term maintenance, and their basic molecular characterization. Further, we spotlight any cell lines that express fluorescent reporters. Additionally, we compare these cell lines with human pluripotent stem cell lines, and we discuss cell lines reprogrammed into a pluripotent naive state, detailing the processes used to attain this. Last, we present the findings from the application of these cell lines in two emerging fields: intra- and interspecies embryonic chimeras and blastoids.

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells.

BACKGROUND: In vitro production of hematopoietic stem/progenitor cells (HSPCs) from human-induced pluripotent stem cells (hiPSCs) provides opportunities for fundamental research, disease modeling, and large-scale production of HLA-matched HSPCs for therapeutic applications. However, a comprehensive understanding of the signaling mechanisms that regulate human hematopoiesis is needed to develop a more effective procedure for deriving HSPCs from hiPSCs. METHODS: In this study, we investigate the role of YAP during the hematopoietic differentiation of hiPSCs to HSPCs and erythrocytes using the isogenic YAP-overexpressing (YAP-S5A) and YAP-depleting (YAP-KD) hiPSCs to eliminate the effects of a genetic background variation. RESULTS: Although YAP is dispensable for maintaining the self-renewal and pluripotency of these hiPSCs, it affects the early cell-fate determination and hematopoietic differentiation of hiPSCs. Depleting YAP enhances the derivation efficiency of HSPCs from hiPSCs by inducing the mesodermal lineage commitment, promoting hematopoietic differentiation, and preventing the differentiation toward endothelial lineage. On the contrary, the overexpression of YAP reduced HSPCs yield by inducing the endodermal lineage commitment, suppressing hematopoietic differentiation, and promoting the differentiation toward endothelial lineage. CONCLUSIONS: Expression of YAP is crucial for the differentiation of hiPSC-derived HSPCs toward mature erythrocytes. We believe that by manipulating YAP activity using small molecules, the efficiency of the large-scale in vitro production system for generating hematopoietic stem/progenitor cells for future therapeutic use could be improved.

Generation of RUNX1c-eGFP induced pluripotent stem cell, MUSIi012-A-4, using CRISPR/Cas9.

Runt-Related Transcription Factor 1c (RUNX1c) plays an important role in regulating the development of hematopoietic stem cells (HSC). Using CRISPR/Cas9 gene editing technology, we established a RUNX1c-eGFP reporter cell line from the MUSIi012-A cell line. The MUSIi012-A-4 cell line has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro and in vivo. This cell line serves as a valuable model to observe the expression of RUNX1c via eGFP tracking during human hematopoietic development.

Derivation of MUSIi016-A iPSCs from peripheral blood with blood type O Rh positive.

MUSIi016-A, a human induced pluripotent stem cell (iPSC), generated from peripheral blood mononuclear cells of a healthy blood group O Rh positive donor was reprogrammed using Sendai viral vectors containing Yamanaka's factors. MUSIi016-A iPSC showed pluripotent stem cell characteristics, highly expressed pluripotent markers, and a capacity to differentiate into all three embryonic cell lineages. This iPSC can be used as a model for the generation of blood cells in vitro.

CRISPR/Cas9 mediated approach to generate YAP-depleted human embryonic stem cell line (MUSIe002-A-1).

Yes-associated protein (YAP), an important effector protein of the Hippo signaling pathway, acts as a molecular switch in controlling cell proliferation and apoptosis. In this study, a YAP-targeted isogenic sub-clone of the MUSIe002-A was generated, designated as MUSIe002-A-1. The MUSIe002-1 cell line had normal pluripotent stem cell characteristics and karyotype. Its ability to differentiate into three germ layers was confirmed. As reduction of YAP does not disturb the pluripotency of hESCs, this cell line serves as a valuable model to extrapolate the functional role of YAP in stem cell biology and its applications.

Episomal vector-based generation of human induced pluripotent stem cell line MUSIi020-A from peripheral blood T-cells.

Human induced pluripotent stem cell (iPSC) line MUSIi020-A was generated from T cells isolated from peripheral blood of a healthy 37-year-old female and reprogrammed using episomal plasmid vectors. The established transgene-free MUSIi020-A, which retained a normal karyotype, displayed pluripotency as characterized by expression of pluripotency markers and by in vitro spontaneous differentiation toward three embryonic germ layers. This cell line may represent a valuable tool for studying T cell development and a potential cell source for cancer immunotherapy.

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton's Jelly Mesenchymal Stem Cells.

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton's jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of ß-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-ß signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.

Derivation of the MUSIe002-A human embryonic stem cell line.

The MUSIe002-A cell line was established from in vitro fertilization of human sperm and oocytes donated for research with informed consent. This cell line exhibited normal human embryonic stem cell (hESC) characteristics, including typical cell morphology, expression of all pluripotent stem cell markers, and potential to differentiate into three germ layers. A karyotyping analysis revealed 46 XY chromosome and cells that did not have mycoplasma contamination. MUSIe002-A represents a valuable unlimited cell source and is of potential interest for human in vitro stem cell based-models, genetic modifications, and stem cell-based therapy of human disease.

Episomal vector reprogramming of human umbilical cord blood natural killer cells to an induced pluripotent stem cell line MUSIi013-A.

Natural killer (NK) cells were isolated from human umbilical cord blood from a healthy newborn and reprogrammed by episomal vectors carrying reprograming factors L-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1, and shRNA against p53 delivered using nucleofection. The obtained MUSIi013-A human induced pluripotent stem cell (iPSC) line highly expressed pluripotency markers, had the capacity to differentiate into derivatives of the three germ layers, while retained a normal karyotype. This cell line may be a useful tool to study epigenic memory that may predispose hiPSCs to enhanced NK differentiation.

Generation of a serine/threonine-protein kinase LATS1 gene-edited iPSC MUSIi012-A-3.

In mammals, there are a number of kinases, including serine/threonine-protein kinase LATS1, that act as a core kinase of the Hippo pathway and that negatively regulate the Hippo effector protein YAP and its paralog TAZ. Using CRISPR/Cas9 technology, we established a stable LATS1 knockdown (LATS1-KD) iPSC from the MUSIi012-A cell line. The LATS1-KD iPSC MUSIi012-A-3 that was developed maintained both the normal karyotype and the pluripotent phenotype, and retained the ability to differentiate into all three embryonic germ layers.

Effect of YAP/TAZ on megakaryocyte differentiation and platelet production.

Platelet transfusion is required for life-threatening thrombocytopenic bleeding, and single donor platelet concentrate is the ideal transfusion product. However, due to the inadequate number of donors that can donate a large volume of platelets, in vitro platelets production could be an alternative. We developed an in vitro production system designed to increase the platelet production yield from cultured cells. Previously, we reported that depletion of a Hippo pathway core kinase (LATS1/2) inhibited platelet production from cultured megakaryocytes. In the present study, we further investigated the role of the Hippo pathway in megakaryocyte proliferation and platelet production by focusing on the role of its effector proteins (YAP and TAZ), which are down-stream targets of LATS1/2 kinase. We found that YAP plays an essential role in megakaryoblastic cell proliferation, maturation, and platelet production, while TAZ showed minor effect. Knockdown of YAP, either by genetic manipulation or pharmaceutical molecule, significantly increased caspase-3-mediated apoptosis in cultured megakaryocytes, and increased platelet production as opposed to overexpressing YAP. We, therefore, demonstrate a paradigm for the regulation of megakaryocyte development and platelet production via the Hippo signaling pathway, and suggest the potential use of an FDA-approved drug to induce higher platelet production in cultured cells.

YAP-depleted iPSC MUSIi012-A-2 maintained all normal stem cell characteristics.

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.

Derivation of human embryonic stem cell line MUSIe001-A from an embryo with homozygous α0-thalassemia (SEA deletion).

MUSIe001-A cell line was derived from a Southeast Asian (SEA) type deletion α0-thalassemia embryo. The SEA deletion embryo was donated for research with informed consent. This cell line shows normal hESC morphology, expresses all pluripotent markers, and has the potential to differentiate into all three germ layers in vitro and in vivo. The MUSIe001-A line has normal karyotype and is free from mycoplasma contamination. PCR analysis confirmed the MUSIe001-A cell line to be a SEA type deletion. MUSIe001-A is a valuable proof of principle model for gene therapy that will facilitate the development of new treatments for affected foetuses.

YAP as a key regulator of adipo-osteogenic differentiation in human MSCs.

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.

Derivation of a MUSIi012-A iPSCs from mobilized peripheral blood stem cells.

CD34+ cells were isolated from mobilized peripheral blood of a healthy donor and reprogrammed by nucleofection with episomal plasmids carrying l-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1, and shRNA against p53. The obtained MUSIi012-A cell line maintained the pluripotent phenotype, the ability to differentiate into all three germ layers, and a normal karyotype.

Generation of human induced pluripotent stem cell line carrying SCN5AC2204>T Brugada mutation (MUSli009-A-1) introduced by CRISPR/Cas9-mediated genome editing.

Human induced pluripotent stem cells (hiPSCs) derived from dermal fibroblasts having wild type (WT) SCN5A were engineered by CRISPR/Cas9-mediated genome editing to harbor a specific point mutation (C2204>T) in SCN5A, which results in a substitution of the WT alanine by valine at codon 735 (A735V). The established MUSli009-A-1 hiPSC line has a homozygous C2204>T mutation on exon 14 of SCN5A that was confirmed by DNA sequencing analysis. The cells exhibited normal karyotype, expressed pluripotent markers and retained its capability to differentiate into three germ layers. The cardiomyocytes derived from this line would be a useful model for investigating cardiac channelopathy.

Generation of a WWTR1 mutation induced pluripotent stem cell line, MUSIi012-A-1, using CRISPR/Cas9.

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.

High-efficiency derivation of human embryonic stem cell lines using a culture system with minimized trophoblast cell proliferation.

BACKGROUND: Due to their extensive self-renewal and multilineage differentiation capacity, human embryonic stem cells (hESCs) have great potential for studying developmental biology, disease modeling, and developing cell replacement therapy. The first hESC line was generated in 1998 by culturing inner cell mass (ICM) cells isolated from human blastocysts using an immunosurgery technique. Since then, many techniques including mechanical ICM isolation, laser dissection, and whole embryo culture have been used to derive hESC lines. However, the hESC derivation efficiency remains low, usually less than 50%, and it requires a large number of human embryos to derive a significant number of hESC lines. Due to a shortage of and restricted access to human embryos, a novel approach with better hESC derivation efficiency is badly needed to decrease the number of embryos used. METHODS: We hypothesized that the low hESC derivation efficiency might be due to extensive proliferation of trophoblast (TE) cells which could interfere with ICM proliferation. We therefore developed a methodology to minimize TE cell proliferation by culturing ICM in a feeder-free system for 3 days before transferring them onto feeder cells. RESULTS: This minimized trophoblast cell proliferation (MTP) technique could be successfully used to derive hESCs from normal, abnormal, and frozen-thawed embryos with better derivation efficiency of more than 50% (range 50-100%; median 70%). CONCLUSIONS: We successfully developed a better hESC derivation methodology using the "MTP" culture system. This methodology can be effectively used to derive hESCs from both normal and abnormal embryos under feeder-free conditions with higher efficiency when compared with other methodologies. With this methodology, large-scale production of clinical-grade hESCs is feasible.

Generation of induced pluripotent stem cells as a potential source of hematopoietic stem cells for transplant in PNH patients.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by lack of CD55 and CD59 on blood cell membrane leading to increased sensitivity of blood cells to complement. Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for PNH, however, lack of HLA-matched donors and post-transplant complications are major concerns. Induced pluripotent stem cells (iPSCs) derived from patients are an attractive source for generating autologous HSCs to avoid adverse effects resulting from allogeneic HSCT. The disease involves only HSCs and their progeny; therefore, other tissues are not affected by the mutation and may be used to produce disease-free autologous HSCs. This study aimed to derive PNH patient-specific iPSCs from human dermal fibroblasts (HDFs), characterize and differentiate to hematopoietic cells using a feeder-free protocol. Analysis of CD55 and CD59 expression was performed before and after reprogramming, and hematopoietic differentiation. Patients' dermal fibroblasts expressed CD55 and CD59 at normal levels and the normal expression remained after reprogramming. The iPSCs derived from PNH patients had typical pluripotent properties and differentiation capacities with normal karyotype. After hematopoietic differentiation, the differentiated cells expressed early hematopoietic markers (CD34 and CD43) with normal CD59 expression. The iPSCs derived from HDFs of PNH patients have normal levels of CD55 and CD59 expression and hold promise as a potential source of HSCs for autologous transplantation to cure PNH patients.