Embryoid Body Formation Research Articles

Reprogramming Prostate Cancer Cells into Induced Pluripotent Stem Cells: a Promising Model of Prostate Cancer Stem Cell Research.

Prostate cancer stem cells (PrCSCs) are responsible for the development of castration-resistant disease and are associated with poor outcomes; however, the origin of PrCSCs is still not known due to the lack of a suitable model. In the current study, the human prostate cancer cell line 22RV1 was used to generate induced pluripotent stem cells (iPSCs) via the exogenous expression of four classic transcription factors (OCT-4, SOX2, KLF4, and C-MYC). The iPSCs were analyzed by phase contrast microscopy, real-time polymerase chain reaction, immunofluorescence, alkaline phosphatase (AP) activity, and examined for karyotype and embryoid body and teratoma formation. The analyses demonstrated that the prostate cancer cells were successfully reprogrammed into iPSCs by characteristic human embryonic stem cell morphology, cell marker expression, AP activity, embryoid body, and pluripotency capability in generating all three embryonic germ layers. These results may provide a convenient and accessible model for studying the origin of PrCSCs and the process by which progenitor cells are transformed into PrCSCs.

Additive manufacturing enables production of de novo cardiomyocytes by controlling embryoid body aggregation

Embryonic stem cells act as a valuable and promising resource in the field of regenerative cell-based therapies and in studying various developmental models. These derived cells can be induced in vitro to differentiate into numerous distinct cell lineages by the formation of Embryoid Bodies (EBs), in a process strongly conditioned by the geometrical characteristics of the EB. This artificial conditioning of cellular differentiation is performed using tools prioritizing geometrical definition but lacking versatility for the adaptation of the geometry to different conditions. Here we demonstrate the production of cardiomyocytes by using high definition direct writing technologies to influence EB aggregation from stem cells. We gauged the impact of this technology over the standard known methods in terms of size dispersion, cell packing density, cardiac tissue health, and the number of cardiomyocytes produced. The feasibility to create small variations of a geometry enabled optimizing EB formation for cardiogenesis and its threefold increase with respect to traditional techniques. This result highlights how the fast-paced improvement of geometrical control in additive manufacture might hold the key to unprecedented control of stem cell differentiation for regenerative medicine.

METTL1-mediated m7G methylation maintains pluripotency in human stem cells and limits mesoderm differentiation and vascular development

Background7-Methylguanosine (m7G) is one of the most conserved modifications in nucleosides within tRNAs and rRNAs. It plays essential roles in the regulation of mRNA export, splicing, and translation. Recent studies highlighted the importance of METTL1-mediated m7G tRNA methylome in the self-renewal of mouse embryonic stem cells (mESCs) through its ability to regulate mRNA translation. However, the exact mechanisms by which METTL1 regulates pluripotency and differentiation in human induced pluripotent stem cells (hiPSCs) remain unknown. In this study, we evaluated the functions and underlying molecular mechanisms of METTL1 in regulating hiPSC self-renewal and differentiation in vivo and in vitro.MethodsBy establishing METTL1 knockdown (KD) hiPSCs, gene expression profiling was performed by RNA sequencing followed by pathway analyses. Anti-m7G northwestern assay was used to identify m7G modifications in tRNAs and mRNAs. Polysome profiling was used to assess the translation efficiency of the major pluripotent transcription factors. Moreover, the in vitro and in vivo differentiation capacities of METTL1-KD hiPSCs were assessed in embryoid body (EB) formation and teratoma formation assays.ResultsMETTL1 silencing resulted in alterations in the global m7G profile in hiPSCs and reduced the translational efficiency of stem cell marker genes. METTL1-KD hiPSCs exhibited reduced pluripotency with slower cell cycling. Moreover, METTL1 silencing accelerates hiPSC differentiation into EBs and promotes the expression of mesoderm-related genes. Similarly, METTL1 knockdown enhances teratoma formation and mesoderm differentiation in vivo by promoting cell proliferation and angiogenesis in nude mice.ConclusionOur findings provided novel insight into the critical role of METTL1-mediated m7G modification in the regulation of hiPSC pluripotency and differentiation, as well as its potential roles in vascular development and the treatment of vascular diseases.

Mthfd2 Modulates Mitochondrial Function and DNA Repair to Maintain the Pluripotency of Mouse Stem Cells.

SummaryThe pluripotency of stem cells determines their developmental potential. While the pluripotency states of pluripotent stem cells are variable and interconvertible, the mechanisms underlying the acquisition and maintenance of pluripotency remain largely elusive. Here, we identified that methylenetetrahydrofolate dehydrogenase (NAD+-dependent), methenyltetrahydrofolate cyclohydrolase (Mthfd2) plays an essential role in maintaining embryonic stem cell pluripotency and promoting complete reprogramming of induced pluripotent stem cells. Mechanistically, in mitochondria, Mthfd2 maintains the integrity of the mitochondrial respiratory chain and prevents mitochondrial dysfunction. In the nucleus, Mthfd2 stabilizes the phosphorylation of EXO1 to support DNA end resection and promote homologous recombination repair. Our results revealed that Mthfd2 is a dual-function factor in determining the pluripotency of pluripotent stem cells through both mitochondrial and nuclear pathways, ultimately ensuring safe application of pluripotent stem cells.

Establishment of a Simple Method for Inducing Neuronal Differentiation of P19 EC Cells without Embryoid Body Formation and Analysis of the Role of Histone Deacetylase 8 Activity in This Differentiation.

P19 pluripotent embryonic carcinoma (EC) stem cells are derived from pluripotent germ cell tumours and can differentiate into three germ layers. Treatment of these cells in suspension culture with retinoic acid induces their differentiation into neurons and glial cells. Hence, these cells are an excellent in vitro model to study the transition from the upper blastoderm to the neuroectoderm. However, because of the complex nature of the techniques involved, the results are highly dependent on the skills of the experimenter. Herein, we developed a simple method to induce neuronal differentiation of adherent P19 EC cells in TaKaRa NDiff® 227 serum-free medium (originally N2B27 medium). This medium markedly induced neuronal differentiation of P19 EC cells. The addition of retinoic acid to the NDiff® 227 medium further enhanced differentiation. Furthermore, cells differentiated by the conventional method, as well as the new method, showed identical expression of the mature neuronal marker, neuronal nuclei. To determine whether our approach could be applied for neuronal studies, we measured histone deacetylase 8 (HDAC8) activity using an HDAC8 inhibitor and HDAC8-knockout P19 EC cells. Inhibition of HDAC8 activity suppressed neuronal maturation. Additionally, HDAC8-knockout cell lines showed immature differentiation compared to the wild-type cell line. These results indicate that HDAC8 directly regulates the neuronal differentiation of P19 EC cells. Thus, our method involving P19 EC cells can be used as an experimental system to study the nervous system. Moreover, this method is suitable for screening drugs that affect the nervous system and cell differentiation.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

SummaryDuchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

BRPF3-HUWE1-mediated regulation of MYST2 is required for differentiation and cell-cycle progression in embryonic stem cells

Brpf-histone acetyltransferase (HAT) complexes have important roles in embryonic development and regulating differentiation in ESCs. Among Brpf family, Brpf3 is a scaffold protein of Myst2 histone acetyltransferase complex that plays crucial roles in gene regulation, DNA replication, development as well as maintaining pluripotency in embryonic stem cells (ESCs). However, its biological functions in ESCs are not elucidated. In this study, we find out that Brpf3 protein level is critical for Myst2 stability and E3 ligase Huwe1 functions as a novel negative regulator of Myst2 via ubiquitin-mediated degradation. Importantly, Brpf3 plays an antagonistic role in Huwe1-mediated degradation of Myst2, suggesting that protein–protein interaction between Brpf3 and Myst2 is required for retaining Myst2 stability. Further, Brpf3 overexpression causes the aberrant upregulation of Myst2 protein levels which in turn induces the dysregulated cell-cycle progression and also delay of early embryonic development processes such as embryoid-body formation and lineage commitment of mouse ESCs. The Brpf3 overexpression-induced phenotypes can be reverted by Huwe1 overexpression. Together, these results may provide novel insights into understanding the functions of Brpf3 in proper differentiation as well as cell-cycle progression of ESCs via regulation of Myst2 stability by obstructing Huwe1-mediated ubiquitination. In addition, we suggest that this is a useful report which sheds light on the function of an unknown gene in ESC field.

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro.

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Negative chronotropic and inotropic effects of lubiprostone on iPS cell-derived cardiomyocytes via activation of CFTR

BackgroundLubiprostone (LBP) is a novel chloride channel opener that has been reported to activate chloride channel protein 2 (ClC-2) and cystic fibrosis transmembrane conductance regulator (CFTR). LBP facilitates fluid secretion by activating CFTR in the intestine and is used as a drug for treating chronic constipation. While ClC-2 and CFTR expression has been confirmed in cardiomyocytes (CMs), the effect of LBP on CMs has not yet been investigated. Thus, the present study aimed to investigate the effect of LBP on CMs using mouse-induced pluripotent stem (iPS) cell-derived CMs (iPS-CMs).MethodsWe induced mouse iPS cells into CMs through embryoid body (EB) formation. We compared the differentiated cells to CMs isolated from adult and fetal mice using gene expression, spontaneous beating rate, and contraction ratio analyses.ResultsGene expression analysis revealed that, in the iPS-CMs, the mRNA expression of the undifferentiated cell markers Rex1 and Nanog decreased, whereas the expression of the unique cardiomyocyte markers cardiac troponin I (cTnI) and cardiac troponin T (cTNT), increased. Immunostaining showed that the localization of cTnI and connexin-43 in the iPS-CMs was similar to that in the primary fetal CMs (FCMs) and adult CMs (ACMs). LBP decreased the spontaneous beating rate of the iPS-CMs and FCMs, and decreased the contraction ratio of the iPS-CMs and ACMs. The reduction in the beating rate and contraction ratio caused by LBP was inhibited by glycine hydrazide (GlyH), which is a CFTR inhibitor.ConclusionThese results suggest that LBP stimulates CFTR in CMs and that LBP has negative chronotropic and inotropic effects on CMs. LBP may be useful for treating cardiac diseases such as heart failure, ischemia, and arrhythmia.

Molecular Mechanisms of Stem Cell Reprogramming by CD47 Antagonists in Primary Human Cells

CD47 is a ubiquitously expressed transmembrane receptor whose interaction with signal regulatory protein alpha on macrophages initiates anti‐phagocytic signaling. Additionally, thrombospondin‐1 (THBS1) binding to CD47 induces cell‐autonomous signaling that regulates cell proliferation, differentiation, and recovery from genotoxic stress. Previous studies showed that CD47‐ or THBS1‐null primary lung endothelial cells exposed to ionizing radiation have higher viability than primary wildtype cells. The mechanisms that enhance the viability of CD47‐null primary cells include enhanced autophagy, anabolic metabolism, and increased expression of c‐Myc and other stem cell transcription factors. The latter enable null cells to spontaneously form embryoid bodies. These results suggest that loss of CD47 may generally facilitate cell reprogramming and self‐renewal. Induced pluripotent stem cells (iPS) can be induced by introducing Yamanaka stem cell factors (MYC, KLF4, SOX2, and OCT4) into primary somatic cells, but iPS cells can form teratomas and thus pose risk of cancer when used for regenerative medicine. Therefore, alternative approaches are needed to overcome these limitations. We propose that CD47 gene knock out (KO) via CRISPR or treating with CD47 antagonists from primary human cells could be a new, simpler, and more effective way to develop multipotent stem cells. To investigate this, we have studied the effects of CD47 knockout and antagonists on embryoid body formation and mRNA expression of stem cell factors using fibroblasts, adipocytes, and human umbilical vein cells (HUVEC). Reprogramming was measured by quantifying embryoid body formation, cell viability, and mRNA expression using real‐time PCR. Preliminary results with HUVEC, fibroblast, and adipocyte primary cells treated with CD47 knockout or antagonists have shown enhanced cell proliferation and expression of stem cell factors similar to murine cd47‐null lung endothelial cells. These results suggest that CD47 plays a key regulatory role in stem cell reprogramming of human primary cells.Support or Funding InformationThis work was supported by the Intramural Research Program of the NIH/National Cancer Institute.

Effect of Ionizing Radiation on Transcriptome during Neural Differentiation of Human Embryonic Stem Cells.

Human embryonic brain development is highly sensitive to ionizing radiation. However, detailed information on the mechanisms of this sensitivity is not available due to limited experimental data. In this study, differentiation of human embryonic stem cells (hESCs) to neural lineages was used as a model for early embryonic brain development to assess the effect of exposure to low (17 mGy) and high (572 mGy) doses of radiation on gene expression. Transcriptomes were assessed using RNA sequencing during neural differentiation at three time points in control and irradiated samples. The first time point was when the cells were still pluripotent (day 0), the second time point was during the stage of embryoid body formation (day 6), and the third and final time point was during the stage of neural rosette formation (day 10). Analysis of the transcriptomes revealed neurodifferentiation in both the control and irradiated cells. Low-dose irradiation did not result in changes in gene expression at any of the time points, whereas high-dose irradiation resulted in downregulation of some major neurodifferentiation markers on days 6 and 10. Gene ontology analysis showed that pathways related to nervous system development, neurogenesis and generation of neurons were among the most affected. Expression of such key regulators of neuronal development as NEUROG1, ARX, ASCL1, RFX4 and INSM1 was reduced more than twofold. In conclusion, exposure to a 17 mGy low dose of radiation was well tolerated by hESCs while exposure to 572 mGy significantly affected their genetic reprogramming into neuronal lineages.

Scalable Generation of Mesenchymal Stem Cells and Adipocytes from Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) can provide unlimited supply for mesenchymal stem cells (MSCs) and adipocytes that can be used for therapeutic applications. Here we developed a simple and highly efficient all-trans-retinoic acid (RA)-based method for generating an off-the-shelf and scalable number of human pluripotent stem cell (hPSC)-derived MSCs with enhanced adipogenic potential. We showed that short exposure of multiple hPSC lines (hESCs/hiPSCs) to 10 μM RA dramatically enhances embryoid body (EB) formation through regulation of genes activating signaling pathways associated with cell proliferation, survival and adhesion, among others. Disruption of cell adhesion induced the subsequent differentiation of the highly expanded RA-derived EB-forming cells into a pure population of multipotent MSCs (up to 1542-fold increase in comparison to RA-untreated counterparts). Interestingly, the RA-derived MSCs displayed enhanced differentiation potential into adipocytes. Thus, these findings present a novel RA-based approach for providing an unlimited source of MSCs and adipocytes that can be used for regenerative medicine, drug screening and disease modeling applications.

Construction of 3D cardiac tissue with synchronous powerful beating using human cardiomyocytes from human iPS cells prepared by a convenient differentiation method

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a new source of cardiac cells are expected to find use as tools in high-throughput screening for drug candidates and cardiotoxicity validation without the need for experimentation on animals. In recent years, it has been reported that drug screening using three-dimensional (3D) tissue is better than conventional 2D culture. Various methods have been developed for mass culture of hiPSC-CMs, and embryoid body (EB) formation is necessary in the majority of differentiation methods as this is reported to promote the differentiation of hiPSCs. However, these operations result in increased processing, cost and loss of hiPSCs. Here, we show alternative methods for differentiation to hiPSC-CMs from <100μm hiPSC-clumps without EB formation and report on a 3D-tissue fabrication using hiPSC-CMs. The 3D cardiac tissue constructed by a layer-by-layer (LbL) cell coating technique (LbL-3D Heart) showed synchronous powerful beating. We conclude that this method enables cost-effective, reproducible and scalable hiPSC-CM production with high activity for tissue engineering, drug screening and regenerative medicine.

Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system.

IntroductionSickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD.ObjectiveGeneration of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium.MethodThis study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed.ResultsThe patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium.ConclusionIn conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

HIF-1α/Actl6a/H3K9ac axis is critical for pluripotency and lineage differentiation of human induced pluripotent stem cells.

Pluripotent stem cells (PSCs) are important models for analyzing cellular metabolism and individual development. As a hypoxia-inducible factor subunit, HIF-1α plays an important role in maintaining the pluripotency of PSCs under hypoxic conditions. However, the mechanisms underlying the self-renewal and pluripotency maintenance of human induced pluripotent stem cells (hiPSCs) via regulating HIF-1α largely remain elusive. In this study, we found that disrupting the expression of HIF-1α reduced self-renewal and pluripotency of hiPSCs. Additionally, HIF-1α-knockdown led to lower mitochondrial membrane potential (ΔΨm ) and higher reactive oxygen species production in hiPSCs. However, HIF-1α-overexpression increased ATP content in hiPSCs, while the role of HIF-1α-knockdown was opposite. The embryoid body (EB) and teratoma formation assays showed that HIF-1α-knockdown promoted endoderm differentiation and development in vitro and in vivo. In terms of the underlying molecular mechanisms, HIF-1α-knockdown inhibited the expression of Actl6a and histone H3K9ac acetylation (H3K9ac). Actl6a knockdown reduced the expression of H3K9ac and the pluripotency of hiPSCs, and also affected endoderm differentiation. These data suggest that hindering HIF-1α expression causes the changes in mitochondrial properties and metabolic disorders in hiPSCs. Furthermore, HIF-1α affects hiPSC pluripotency, and germ layer differentiation via Actl6a and histone acetylation.

Generation of induced pluripotent stem cell line RCPCMi004-A derived from patient with Parkinson's disease with deletion of the exon 2 in PARK2 gene.

IPSC line RCPCMi004-A was generated from skin fibroblasts collected from a male patient with early onset Parkinson's disease. The patient carries a heterozygous deletion of the exon 2 of PARK2 gene. The reprogramming of fibroblasts was performed with Sendai viruses containing Oct-4, Sox-2, Klf-4 and c-Myc. Pluripotency was confirmed by immunofluorescence, RT-PCR, and formation of embryoid bodies. The RCPCMi004-A cell line carries the same deletion in PARK2 gene. The RCPCMi004-A cell line can be used to model Parkinson's disease in vitro.

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

The role of Hippo signaling pathway and mechanotransduction in tuning embryoid body formation and differentiation.

Embryoid bodies (EBs) are the three-dimensional aggregates of pluripotent stem cells that are used as a model system for the in vitro differentiation. EBs mimic the early stages of embryogenesis and are considered as a potential biomimetic body in tuning the stem cell fate. Although EBs have a spheroid shape, they are not formed accidentally by the agglomeration of cells; they are formed by the deliberate and programmed aggregation of stem cells in a complex topological and biophysical microstructure instead. EBs could be programmed to promisingly differentiate into the desired germ layers with specific cell lineages, in response to intra- and extra-biochemical and biomechanical signals. Hippo signaling and mechanotransduction are the key pathways in controlling the formation and differentiation of EBs. The activity of the Hippo pathway strongly relies on cell-cell junctions, cell polarity, cellular architecture, cellular metabolism, and mechanical cues in the surrounding microenvironment. Although the Hippo pathway was initially thought to limit the size of the organ by inhibiting the proliferation and the promotion of apoptosis, the evidence suggests that this pathway even regulates stem cell self-renewal and differentiation. Considering the abovementioned explanations, the present study investigated the interplay of the Hippo signaling pathway, mechanotransduction, differentiation, and proliferation pathways to draw the molecular network involved in the control of EBs fate. In addition, this study highlighted several neglected critical parameters regarding EB formation, in the interplay with the Hippo core component involved in the promising differentiation.

Haspin-dependent and independent effects of the kinase inhibitor 5-Iodotubercidin on self-renewal and differentiation

The kinase Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), creating a docking site for the Chromosomal Passenger Complex (CPC). CPC plays a pivotal role in preventing chromosome misalignment. Here, we have examined the effects of 5-Iodotubercidin (5-ITu), a commonly used Haspin inhibitor, on self-renewal and differentiation of mouse embryonic stem cells (ESCs). Treatment with low concentrations of 5-ITu eliminates the H3T3ph mark during mitosis, but does not affect the mode or the outcome of self-renewal divisions. Interestingly, 5-ITu causes sustained accumulation of p53, increases markedly the expression of histone genes and results in reversible upregulation of the pluripotency factor Klf4. However, the properties of 5-ITu treated cells are distinct from those observed in Haspin-knockout cells generated by CRISPR/Cas9 genome editing, suggesting “off-target” effects. Continuous exposure to 5-ITu allows modest expansion of the ESC population and growth of embryoid bodies, but release from the drug after an initial treatment aborts embryoid body or teratoma formation. The data reveal an unusual robustness of ESCs against mitotic perturbants and suggest that the lack of H3T3ph and the “off-target” effects of 5-ITu can be partially compensated by changes in expression program or accumulation of suppressor mutations.