Efficiency Of iPSC Generation Research Articles

Inducing Pluripotency in Somatic Cells: Historical Perspective and Recent Advances.

Inducing pluripotency in somatic cells is mediated by the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc. The resulting induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine by virtue of their ability to differentiate into different types of functional cells. Specifically, iPSCs derived directly from patients offer a powerful platform for creating in vitro disease models. This facilitates elucidation of pathological mechanisms underlying human diseases and development of new therapeutic agents mitigating disease phenotypes. Furthermore, genetically and phenotypically corrected patient-derived iPSCs by gene-editing technology or the supply of specific pharmaceutical agents can be used for preclinical and clinical trials to investigate their therapeutic potential. Despite great advances in developing reprogramming methods, the efficiency of iPSC generation remains still low and varies between donor cell types, hampering the potential application of iPSC technology. This paper reviews histological timeline showing important discoveries that have led to iPSC generation and discusses recent advances in iPSC technology by highlighting donor cell types employed for iPSC generation.

Hepatitis B Virus x Protein Increases Cellular OCT3/4 and MYC and Facilitates Cellular Reprogramming.

Hepatitis B virus x (HBx) is a multifunctional protein coded by the Hepatitis B virus that is involved in various cellular processes such as proliferation, cell survival/apoptosis, and histone methylation. HBx was reported to be associated with liver "cancer stem cells." The stemness inducing properties of HBx could also facilitate the generation of pluripotent stem cells from somatic cells. It is well established that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) using a cocktail of transcription factors called Yamanaka's factors (YFs) (OCT4, SOX2, KLF4, and MYC). The reprogramming process proceeds step-by-step with reprogramming factor chromatin interactions, transcription, and chromatin states changing during transitions. HBx is a "broad spectrum trans-activator" and therefore could facilitate these transitions. We electroporated low passage and high passage (difficult to reprogram) fibroblasts using YFs with and without HBx and evaluated the reprogramming efficiency. We also investigated the tri-lineage and terminal differentiation potential of iPSC derived using HBx. We found that the addition of HBx to YF improves iPSC derivation, and it increases the efficiency of iPSC generation from "difficult or hard-to-reprogram samples" such as high passage/senescent fibroblasts. Further, we show that HBx can substitute the key transcription factor MYC in the YF cocktail to generate iPSC. The cellular levels of OCT3/4 and MYC were increased in HBx expressing cells. Our results have practical value in improving the efficiency of pluripotent stem cell derivation from "difficult to reprogram" somatic cells, in addition to providing some insights into the mechanisms of liver carcinogenesis in chronic hepatitis B. To conclude, HBx improves the reprogramming efficiency of YFs. HBx increases the cellular levels of OCT3/4 and MYC.

Tryptophan Hydroxylase-2-Mediated Serotonin Biosynthesis Suppresses Cell Reprogramming into Pluripotent State.

The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.

Enhanced efficiency of generating human-induced pluripotent stem cells using Lin28-30Kc19 fusion protein.

Induced pluripotent stem cells (iPSCs) have intrinsic properties, such as self-renewal ability and pluripotency, which are also shown in embryonic stem cells (ESCs). The challenge of improving the iPSC generation efficiency has been an important issue and there have been many attempts to develop iPSC generation methods. In this research, we added Lin28, known as one of the reprogramming factors, in the form of a soluble recombinant protein from E. coli to improve the efficiency of human iPSC (hiPSC) generation, in respect of alkaline phosphatase (AP)-positive colonies. To deliver Lin28 inside the cells, we generated a soluble Lin28-30Kc19 fusion protein, with 30Kc19 at the C-terminal domain of Lin28. 30Kc19, a silkworm hemolymph-derived protein, was fused due to its cell-penetrating and protein-stabilizing properties. The Lin28-30Kc19 was treated to human dermal fibroblasts (HDFs), in combination with four defined reprogramming factors (Oct4, Sox2, c-Myc, and Klf4). After 14 days of cell culture, we confirmed the generated hiPSCs through AP staining. According to the results, the addition of Lin28-30Kc19 increased the number and size of generated AP-positive hiPSC colonies. Through this research, we anticipate that this recombinant protein would be a valuable material for increasing the efficiency of hiPSC generation and for enhancing the possibility as a substitute of the conventional method.

DNA Damage Response and Cell Cycle Regulation in Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy because of their pluripotent property and the ability to proliferate indefinitely. Embryonic stem cells (ESCs) derived from inner cell mass (ICM) possess unique cell cycle control with shortened G1 phase. In addition, ESCs have high expression of homologous recombination (HR)-related proteins, which repair double-strand breaks (DSBs) through HR or the non-homologous end joining (NHEJ) pathway. On the other hand, the generation of induced pluripotent stem cells (iPSCs) by forced expression of transcription factors (Oct4, Sox2, Klf4, c-Myc) is accompanied by oxidative stress and DNA damage. The DNA repair mechanism of DSBs is therefore critical in determining the genomic stability and efficiency of iPSCs generation. Maintaining genomic stability in PSCs plays a pivotal role in the proliferation and pluripotency of PSCs. In terms of therapeutic application, genomic stability is the key to reducing the risks of cancer development due to abnormal cell replication. Over the years, we and other groups have identified important regulators of DNA damage response in PSCs, including FOXM1, SIRT1 and PUMA. They function through transcription regulation of downstream targets (P53, CDK1) that are involved in cell cycle regulations. Here, we review the fundamental links between the PSC-specific HR process and DNA damage response, with a focus on the roles of FOXM1 and SIRT1 on maintaining genomic integrity.

Valproic Acid Enhance Reprogramming of Bactrian Camel Cells through Promoting the Expression of Endogenous Gene c-Myc and the Process of Angiogenesis.

Background and ObjectivesInduced pluripotent stem cells (iPSCs) are usually generated by reprogramming differentiated cells through the introduction of specific transcription factors, but this is a difficult and inefficient process. Valproic acid (VPA) is a histone deacetylase inhibitor that significantly improves the efficiency of iPSC generation. But its role and mechanism are still unclear.Methods and ResultsWe transduced Bactrian camel fetal fibroblasts (BCFFs) with retroviruses carrying defined factors (OCT4, SOX2, KLF4, c-MYC and EGFP; OSKMG) in the presence of VPA. Cells were collected (Day 7) and analyzed using RNA-seq technology. Afterwards, different groups of cells and transcriptomics results were detected by PCR and qRT-PCR technology. The results showed that VPA promoted the expression of the endogenous gene c-Myc and inhibited cell proliferation; at the same time, it promoted the expression of VEGF and other genes related to angiogenesis.ConclusionsWhen VPA is added to the culture medium, only the cells that have begun to reprogram can break the G2/M repression through the expression of the endogenous gene c-Myc, and use the nutrients and space in the culture dish to proliferate normally, which can achieve the purpose of directly improving the efficiency of reprogramming. Another new discovery for Bactrian camels, VPA significantly increased the expression of VEGFC and other genes, promoting the transformation of fibroblasts to endothelial cells (different from the mesenchymal-to-epithelial transition process of other species) to accelerate the early induction of Bactrian camels iPSc process. Overall, this study proved the new mechanism of VPA in enhancing the induction of pluripotency from the transcriptome level.

Rad50 mediates DNA demethylation to establish pluripotent reprogramming

DNA demethylation is characterized by the loss of methyl groups from 5-methylcytosine, and this activity is involved in various biological processes in mammalian cell development and differentiation. In particular, dynamic DNA demethylation in the process of somatic cell reprogramming is required for successful iPSC generation. In the present study, we reported the role of Rad50 in the DNA demethylation process during somatic cell reprogramming. We found that Rad50 was highly expressed in pluripotent stem cells and that Rad50 regulated global DNA demethylation levels. Importantly, the overexpression of Rad50 resulted in the enhanced efficiency of iPSC generation via increased DNA demethylation, whereas Rad50 knockdown led to DNA hypermethylation, which suppressed somatic cell reprogramming into iPSCs. Moreover, we found that Rad50 associated with Tet1 to facilitate the DNA demethylation process in pluripotent reprogramming. Therefore, our findings highlight the novel role of Rad50 in the DNA demethylation process during somatic cell reprogramming.

Absence of cyclin-dependent kinase inhibitor p27 or p18 increases efficiency of iPSC generation without induction of iPSC genomic instability

Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.

Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed.In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction.Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%).Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. [Display omitted] DisclosuresLiao:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

Optimization of reprogramming culture conditions for the generation of induced pluripotent stem cells from Col1a1 4F2A-Oct4-GFP mice with high efficiency.

A reprogrammable transgenic mouse strain, called Col1a1 4F2A-Oct4-GFP, was bred for the present study. Because the somatic cells of this mouse strain contain only two copies of each Yamanaka factor, these animals are inefficient at producing induced pluripotent stem cells (iPSCs; approx. 0.005%) under traditional culture conditions. With an optimized culture condition, the iPSC production rate of mouse embryonic fibroblasts (MEFs) of Col1a1 4F2A-Oct4-GFP mice (MEFCol1a14F2A-Oct4-GFP ) was increased to approximately 8%. Further, promotion of cell proliferation by serum supplementation did not enhance iPSC production. Inhibition of transforming growth factor β (TGF-β) in the serum by SB431542 neither affected the growth rate of MEFCol1a14F2A-Oct4-GFP nor promoted iPSC production. However, the use of the gamma-irradiated STO-NEO-LIF (γSNL) cells to serve as feeders for iPSC production resulted in a 5-fold higher rate of iPSC production than the use of γMEFICR feeders. Interestingly, the use of SB431542 with the γMEFICR -adopted system could eliminate this difference. RT-PCR-based comparative analysis further demonstrated that TGF-β expression was 10-fold higher in γMEFICR than in γSNL cells. Consistent with previous reports, mesenchymal to epithelial transition was found to participate in the initial steps of reprogramming in the specific context of MEFCol1a14F2A-Oct4-GFP . Moreover, we found that the initial seeding density is one of the pivotal factors for determining a high efficiency of iPSC generation. The iPSCs efficiently generated from our MEFCol1a14F2A-Oct4-GFP resembled mouse embryonic stem cells (mESCs) in aspects of teratoma formation and germline transmission. Depending on the culture conditions, our Col1a1 4F2A-Oct4-GFP mouse system can generate bona fide iPSCs with variable efficiencies, which can serve as a tool for interrogating the route taken by cells during somatic reprogramming.

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.

Enhanced generation of iPSCs from older adult human cells by a synthetic five-factor self-replicative RNA

We previously devised a polycistronic, synthetic self-replicating RNA (srRNA) to generate human induced Pluripotent Stem Cells (iPSCs) that simultaneously expresses four reprogramming factors (4F). However, while the best 4F srRNA efficiently generated iPSCs from young fibroblasts, it was inefficient on adult human fibroblasts (>50 years). To increase the iPSC generation efficiency, we included additional reprogramming factors. We found that a single transfection of a five factor (5F) srRNA, containing OCT4, KLF4, SOX2, GLIS1 and c-MYC, robustly generated iPSCs from adult human fibroblasts aged 54 to 77 and from a 24 year old cardiomyopathy patient donor. Interestingly, 5F-srRNA induced LIN28A, which was one of the original reprogramming factors. 5F-srRNA also accelerated the generation of iPSCs by seven days compared to 4F-srRNAs. Further improvements include phosphatase treatment to remove 5' phosphate and use of Lipofectamine MessengerMAX that increased transfection efficiency to ~90%. Together, these improvements enabled us to efficiently generate iPSCs from human fibroblasts using 5F-srRNA while eliminating both puromycin selection and feeder cells.

Genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1) improves cell reprogramming efficiency and increases induced pluripotent stem cell proliferation and survival

Adult fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSC) for use in various applications. However, there are challenges in iPSC generation including low reprogramming efficiency, yield, cell survival and viability. Since the Hippo signalling pathway is a key pathway involved in regulating cell proliferation and survival, we here test whether modification of the Hippo pathway will enhance the efficiency of iPSC generation and improve their survival.The Hippo pathway was modified by genetic ablation of the mammalian sterile-20 like kinase 1 (Mst1), a major component of the pathway. Using adult skin fibroblasts isolated from Mst1 knockout mice (Mst1−/−) as a source of iPSC we found that genetic ablation of Mst1 leads to significantly increased reprogramming efficiency by 43.8%. Moreover, Mst1−/− iPSC displayed increase proliferation by 12% as well as an increase in cell viability by 20% when treated with a chemical hypoxic inducer. Mechanistically, we found higher activity of YAP, the main downstream effector of the Hippo pathway, in iPSC lacking Mst1.In conclusion, our data suggests that Mst1 can be targeted to improve the efficiency of adult somatic cell reprogramming as well as to enhance iPSC proliferation and survival.

RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by miRNA-Based Repression of RYBP and its PRC1-Dependent and -Independent Functions

RNA-binding proteins (RBPs), in addition to their functions in cellular homeostasis, play important roles in lineage specification and maintaining cellular identity. Despite their diverse and essential functions, which touch on nearly all aspects of RNA metabolism, the roles of RBPs in somatic cell reprogramming are poorly understood. Here we show that the DEAD-box RBP DDX5 inhibits reprogramming by repressing the expression and function of the non-canonical polycomb complex 1 (PRC1) subunit RYBP. Disrupting Ddx5 expression improves the efficiency of iPSC generation and impedes processing of miR-125b, leading to Rybp upregulation and suppression of lineage-specific genes via RYBP-dependent ubiquitination of H2AK119. Furthermore, RYBP is required for PRC1-independent recruitment of OCT4 to the promoter of Kdm2b, a histone demethylase gene that promotes reprogramming by reactivating endogenous pluripotency genes. Together, these results reveal important functions of DDX5 in regulating reprogramming and highlight the importance of a Ddx5-miR125b-Rybp axis in controlling cell fate.

Human Mutations Affecting Reprogramming into Induced Pluripotent Stem Cells

The development of induced pluripotent stem cells (iPSCs) provides unprecedented opportunities for life sciences, drug discovery, and regenerative medicine. iPSCs have been generated from somatic cells in many patients with various genetic diseases carrying specific mutations. However, the efficiency of iPSC generation is quite low. Less than 1% of human primary somatic cells can usually turn into iPSCs. Previous studies have revealed that cellular signaling pathways, epigenetic status, and cellular senescence were major barriers to iPSC generation. Serendipitously in some cases, human mutations themselves affect the reprogramming efficiency of iPSC generation as well as cellular phenotypes recapitulating their disease symptoms. Mutations, which cause altered DNA repair (e.g., Ataxia-Telangiectasia, fanconi anemia and DNA Ligase IV (LIG4) syndrome), premature aging (e.g., Hutchinson–Gilford progeria syndrome and Nestor–Guillermo progeria syndrome), altered telomere homeostasis (e.g., dyskeratosis congenita), mitochondrial respiratory dysfunction, chromosomal abnormalities, and fibrodysplasia ossificans progressiva, have all been shown to affect the reprogramming efficiency of somatic cells to iPSCs. In this review, the effects of such mutations are summarized and the methods which have been employed to rescue efficient iPSC generation from mutant cells is discussed. Although the mutations affecting reprogramming processes are rare, these mutations have been invaluable to the elucidation of reprogramming mechanisms and to the development of improved reprogramming technologies.

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts

Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

H1foo Has a Pivotal Role in Qualifying Induced Pluripotent Stem Cells.

SummaryEmbryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.

Histone chaperone APLF regulates induction of pluripotency in murine fibroblasts.

Induction of pluripotency in differentiated cells through the exogenous expression of the transcription factors Oct4, Sox2, Klf4 and cellular Myc involves reprogramming at the epigenetic level. Histones and their metabolism governed by histone chaperones constitute an important regulator of epigenetic control. We hypothesized that histone chaperones facilitate or inhibit the course of reprogramming. For the first time, we report here that the downregulation of histone chaperone Aprataxin PNK-like factor (APLF) promotes reprogramming by augmenting the expression of E-cadherin (Cdh1), which is implicated in the mesenchymal-to-epithelial transition (MET) involved in the generation of induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Downregulation of APLF in MEFs expedites the loss of the repressive MacroH2A.1 (encoded by H2afy) histone variant from the Cdh1 promoter and enhances the incorporation of active histone H3me2K4 marks at the promoters of the pluripotency genes Nanog and Klf4, thereby accelerating the process of cellular reprogramming and increasing the efficiency of iPSC generation. We demonstrate a new histone chaperone (APLF)-MET-histone modification cohort that functions in the induction of pluripotency in fibroblasts. This regulatory axis might provide new mechanistic insights into perspectives of epigenetic regulation involved in cancer metastasis.

Improvement in Mouse iPSC Induction by Rab32 Reveals the Importance of Lipid Metabolism during Reprogramming

Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages, and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency, and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.