Blockage of retinoic acid signaling via RARγ suppressed the proliferation of pancreatic cancer cells by arresting the cell cycle progression of the G1-S phase.

BACKGROUND: Our study and several studies have reported that in some cancers, including pancreatic ductal adenocarcinoma (PDAC), the expression of squamous lineage markers, such as esophagus-tissue-specific genes, correlated with a poor prognosis. However, the mechanism by which the acquisition of squamous lineage phenotypes leads to a poor prognosis remains unclear. We previously reported that retinoic acid signaling via retinoic acid receptor γ (RARγ signaling) determines the differentiation lineage into the esophageal squamous epithelium. These findings hypothesized that the activation of RARγ signaling contributed to acquiring squamous lineage phenotypes and malignant behavior in PDAC. METHODS: This study utilized public databases and immunostaining of surgical specimens to examine RARγ expression in PDAC. We evaluated the function of RARγ signaling by inhibitors and siRNA knockdown using a PDAC cell line and patient-derived PDAC organoids. The mechanism of the tumor-suppressive effects by blocking RARγ signaling was examined by a cell cycle analysis, apoptosis assays, RNA sequencing and Western blotting. RESULTS: RARγ expression in pancreatic intraepithelial neoplasia (PanIN) and PDAC was higher than that in the normal pancreatic duct. Its expression correlated with a poor patient prognosis in PDAC. In PDAC cell lines, blockade of RARγ signaling suppressed cell proliferation by inducing cell cycle arrest in the G1 phase without causing apoptosis. We demonstrated that blocking RARγ signaling upregulated p21 and p27 and downregulated many cell cycle genes, including cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6. Furthermore, using patient-derived PDAC organoids, we confirmed the tumor-suppressive effect of RARγ inhibition and indicated the synergistic effects of RARγ inhibition with gemcitabine. CONCLUSIONS: This study clarified the function of RARγ signaling in PDAC progression and demonstrated the tumor-suppressive effect of selective blockade of RARγ signaling against PDAC. These results suggest that RARγ signaling might be a new therapeutic target for PDAC.

Re-generation of cytotoxic γδT cells with distinctive signatures from human γδT-derived iPSCs.

For a long time, ex vivo-expanded peripheral-blood-derived γδT cell (PBγδT)-based immunotherapy has been attractive, and clinical trials have been undertaken. However, the difficulty in expanding cytotoxic γδT cells to an adequate number has been a major limitation to the efficacy of treatment in most cases. We successfully re-generated γδT cells from γδT cell-derived human induced pluripotent stem cells (iPSCs). The iPSC-derived γδT cells (iγδTs) killed several cancer types in a major histocompatibility complex (MHC)-unrestricted manner. Single-cell RNA sequencing (scRNA-seq) revealed that the iγδTs were identical to a minor subset of PBγδTs. Compared with a major subset of PBγδTs, the iγδTs showed a distinctive gene expression pattern: lower CD2, CD5, and antigen-presenting genes; higher CD7, KIT, and natural killer (NK) cell markers. The iγδTs expressed granzyme B and perforin but not interferon gamma (IFNγ). Our data provide a new source for γδT cell-based immunotherapy without quantitative limitation.

Chorioallantoic membrane assay revealed the role of TIPARP (2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible poly (ADP-ribose) polymerase) in lung adenocarcinoma-induced angiogenesis.

BACKGROUND: The chorioallantoic membrane (CAM) assay is a well-established technique to evaluate tumor invasion and angiogenesis and may overcome the shortcoming of the patient-derived xenograft (PDX) mouse model. Currently, few reports have described lung cancer invasion and angiogenesis in the CAM assay. We therefore used the CAM assay in the evaluation of lung cancer. METHOD: Lung cancer cell line-derived organoids or lung cancer cell lines were transplanted into the CAM on embryonic development day (EDD) 10, and an analysis was performed on EDD 15. Microscopic and macroscopic images and movies of the grafts on the CAM were captured and analyzed. The relationships between the graft and chick vessels were evaluated using immunohistochemistry. RESULTS: We transplanted lung cancer cell lines and cell line-derived organoid into a CAM to investigate angiogenesis and invasion. They engrafted on the CAM at a rate of 50-83%. A549-OKS cells showed enhanced cell invasion and angiogenesis on the CAM in comparison to A549-GFP cells as was reported in vitro. Next, we found that A549-TIPARP cells promoted angiogenesis on the CAM. RNA-seq identified 203 genes that were upregulated more than twofold in comparison to A549-GFP cells. A pathway analysis revealed many upregulated pathways related to degradation and synthesis of the extracellular matrix in A549-TIPARP cells. CONCLUSIONS: The CAM assay can be used to evaluate and research invasion and angiogenesis in lung cancer. The elevated expression of TIPARP in lung cancer may induce angiogenesis by remodeling the extracellular matrix.

Identification and characterization of slow­cycling cells in Ewing sarcoma.

Ewing sarcoma (ES) is an aggressive primary malignant bone tumor that predominantly affects children and young adults. Multimodal treatment approaches have markedly improved the survival of patients with localized ES. However, local recurrence and distant metastasis following curative therapies remain a main concern for patients with ES. Recent studies have suggested that slow­cycling cells (SCCs) are associated with tumor progression, local recurrence and distant metastasis in various types of cancers. According to the results of these studies, it was hypothesized that SCCs may play a critical role in tumor progression, chemoresistance and local/distal recurrence in patients with ES. The present study applied a label­retaining system using carboxyfluorescein diacetate succinimidyl ester (CFSE) to identify and isolate SCCs in ES cell lines. In addition, the properties of SCCs, including sphere formation ability, cell cycle distribution and chemoresistance, in comparison with non­SCCs were investigated. RNA sequencing also revealed several upregulated genes in SCCs as compared with non­SCCs; the identified genes not only inhibited cell cycle progression, but also promoted the malignant properties of SCCs. On the whole, the present study successfully identified SCCs in ES cells through a label­retaining system using CFSE. Moreover, to the best of our knowledge, the present study is the first to describe the characteristic properties of SCCs in ES. The findings of this study, if confirmed, may prove to be useful in elucidating the underlying molecular mechanisms and identifying effective therapeutic targets for ES.

CDX2-induced intestinal metaplasia in human gastric organoids derived from induced pluripotent stem cells.

Intestinal metaplasia is related to gastric carcinogenesis. Previous studies have suggested the important role of CDX2 in intestinal metaplasia, and several reports have shown that the overexpression of CDX2 in mouse gastric mucosa caused intestinal metaplasia. However, no study has examined the induction of intestinal metaplasia using human gastric mucosa. In the present study, to produce an intestinal metaplasia model in human gastric mucosa in vitro, we differentiated human-induced pluripotent stem cells (hiPSC) to gastric organoids, followed by the overexpression of CDX2 using a tet-on system. The overexpression of CDX2 induced, although not completely, intestinal phenotypes and the enhanced expression of many, but not all, intestinal genes and previously reported intestinal metaplasia-related genes in the gastric organoids. This model can help clarify the mechanisms underlying intestinal metaplasia and carcinogenesis in human gastric mucosa and develop therapies to restitute precursor conditions of gastric cancer to normal mucosa.

Increased expression of SPRR1A is associated with a poor prognosis in pancreatic ductal adenocarcinoma.

OBJECTIVES: Small proline-rich protein 1A (SPRR1A) is recognized as a squamous differentiation marker but is also upregulated in some non-squamous cancers. However, its expression in pancreatic ductal adenocarcinoma (PDAC) has not been investigated. This study elucidated the expression of SPRR1A in PDAC and its effect on the prognosis and malignant behavior of PDAC. METHODS: We examined the SPRR1A expression by immunohistochemistry in 86 surgical PDAC cases and revealed the relationship between its expression and the prognosis of the PDAC patients. Furthermore, we overexpressed SPRR1A in pancreatic cancer cell lines (PK-1 and Panc-1) and assessed the phenotype and gene expression changes in vitro. RESULTS: Among the 84 cases, excluding 2 with squamous differentiation, 31 (36.9%) had a high SPRR1A expression. The overall survival (median 22.1 months vs. 33.6 months, p = 0.0357) and recurrence-free survival (median 10.7 months vs. 15.5 months, p = 0.0298) were significantly lower in the high-SPRR1A-expression group than in the low-SPRR1A-expression group. A multivariate analysis indicated that a high SPRR1A expression (HR 1.706, 95% CI 1.018 to 2.862, p = 0.0427) and residual tumor status (HR 2.687, 95% CI 1.487 to 4.855, p = 0.00106) were independent prognostic factors. The analysis of TCGA transcriptome data demonstrated that the high-SPRR1A-expression group had a significantly worse prognosis than the low-SPRR1A-expression group, which supported our data. SPRR1A overexpression in PK-1 and Panc-1 did not result in remarkable changes to in vitro phenotypes, such as the cell proliferation, chemo-resistance, EMT, migration or global gene expression. CONCLUSION: Increased expression of SPRR1A is associated with a poor prognosis in PDAC and may serve as a novel prognostic marker. However, our in vitro study suggests that the SPRR1A expression may be a consequence, not a cause, of the aggressive behavior of PDAC.

Epithelial-derived factors induce muscularis mucosa of human induced pluripotent stem cell-derived gastric organoids.

Human gastric development has not been well studied. The generation of human pluripotent stem cell-derived gastric organoids (hGOs) comprising gastric marker-expressing epithelium without an apparent smooth muscle (SM) structure has been reported. We modified previously reported protocols to generate hGOs with muscularis mucosa (MM) from hiPSCs. Time course analyses revealed that epithelium development occurred prior to MM formation. Sonic hedgehog (SHH) and TGF-ß1 were secreted by the epithelium. HH and TGF-ß signal inhibition prevented subepithelial MM formation. A mechanical property of the substrate promoted SM differentiation around hGOs in the presence of TGF-ß. TGF-ß signaling was shown to influence the HH signaling and mechanical properties. In addition, clinical specimen findings suggested the involvement of TGF-ß signaling in MM formation in recovering gastric ulcers. HH and TGF-ß signaling from the epithelium to the stroma and the mechanical properties of the subepithelial environment may influence the emergence of MM in human stomach tissue.

Restoration of the defect in radial glial fiber migration and cortical plate organization in a brain organoid model of Fukuyama muscular dystrophy.

Fukuyama congenital muscular dystrophy (FCMD) is a severe, intractable genetic disease that affects the skeletal muscle, eyes, and brain and is attributed to a defect in alpha dystroglycan (αDG) O-mannosyl glycosylation. We previously established disease models of FCMD; however, they did not fully recapitulate the phenotypes observed in human patients. In this study, we generated induced pluripotent stem cells (iPSCs) from a human FCMD patient and differentiated these cells into three-dimensional brain organoids and skeletal muscle. The brain organoids successfully mimicked patient phenotypes not reliably reproduced by existing models, including decreased αDG glycosylation and abnormal radial glial (RG) fiber migration. The basic polycyclic compound Mannan-007 (Mn007) restored αDG glycosylation in the brain and muscle models tested and partially rescued the abnormal RG fiber migration observed in cortical organoids. Therefore, our study underscores the importance of αDG O-mannosyl glycans for normal RG fiber architecture and proper neuronal migration in corticogenesis.

Differentiation of Human Induced Pluripotent Stem Cells Into Testosterone-Producing Leydig-like Cells.

Late-onset hypogonadism (LOH) syndrome, due to a partial lack of testosterone, decreases the quality of life of older men. Testosterone is mainly secreted by Leydig cells in the testes. Leydig cell transplantation is expected to be a promising alternative to conventional testosterone replacement therapy for LOH syndrome. We herein report a simple and robust protocol for directed differentiation of human induced pluripotent stem cells (hiPSCs) into Leydig-like cells by doxycycline-inducible overexpression of NR5A1 and treatment with a combination of 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) and forskolin. The differentiated cells expressed the steroidogenic enzyme genes STAR, CYP11A1, CYP17A1, and HSD3B2 and the specific markers of adult Leydig cells HSD17B3, INSL3, and LHCGR. Furthermore, we confirmed the secretion of functional testosterone from the cells into the culture supernatant by a testosterone-sensitive cell proliferation assay. These findings showed that the hiPSCs were able to be differentiated into Leydig-like cells, supporting the expectation that hiPSC-derived Leydig-like cells can be novel tools for treating LOH syndrome.

Acquisition of cancer stem cell properties in osteosarcoma cells by defined factors.

BACKGROUND: Cancer stem cells (CSCs) are considered to be responsible for tumor initiation, formation, and poor prognosis of cancer patients. However, the rarity of CSCs in clinical samples makes it difficult to elucidate characteristics of CSCs, especially in osteosarcoma (OS). The aim of this study is to verify whether it is possible to generate CSC-like cells by transducing defined factors into an OS cell line. METHODS: We retrovirally transduced the Octamer-binding transcription factor 3/4 (OCT3/4), Kruppel-like factor 4 (KLF4), and SRY-box transcription factor 2 (SOX2) genes into the MG-63 human OS cell line (MG-OKS). Parental and GFP-transduced MG-63 cells were used as negative control. We assessed the properties of the generated cells in vitro and in vivo. Multiple comparisons among groups were made using a one-way analysis of variance (ANOVA) followed by post hoc testing with Tukey's procedure. RESULTS: MG-OKS cells in vitro exhibited the significantly increased mRNA expression levels of CSC markers (CD24, CD26, and CD133), decreased cell growth, increased chemoresistance and cell migration, and enhanced sphere formation. Notably, MG-OKS cells cultured under osteogenic differentiation conditions showed strongly positive staining for both Alizarin Red S and alkaline phosphatase, indicating osteogenesis of the cells. Gene ontology analysis of microarray data revealed significant upregulation of epidermal-related genes. Tumors derived from MG-OKS cells in vivo were significantly larger than those from other cells in µCT analysis, and immunohistochemical staining showed that Ki-67, osteocalcin, and HIF-1α-positive cells were more frequently detected in the MG-OKS-derived tumors. CONCLUSIONS: In this study, we successfully generated OS CSC-like cells with significantly enhanced CSC properties following transduction of defined factors.

Correction to: Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium.

In the original publication of the article, the following errors were noted and corrected in this correction.

Retinoic acid receptor γ activation promotes differentiation of human induced pluripotent stem cells into esophageal epithelium.

BACKGROUND: The esophagus is known to be derived from the foregut. However, the mechanisms regulating this process remain unclear. In particular, the details of the human esophagus itself have been poorly researched. In this decade, studies using human induced pluripotent stem cells (hiPSCs) have proven powerful tools for clarifying the developmental biology of various human organs. Several studies using hiPSCs have demonstrated that retinoic acid (RA) signaling promotes the differentiation of foregut into tissues such as lung and pancreas. However, the effect of RA signaling on the differentiation of foregut into esophagus remains unclear. METHODS: We established a novel stepwise protocol with transwell culture and an air-liquid interface system for esophageal epithelial cell (EEC) differentiation from hiPSCs. We then evaluated the effect of all-trans retinoic acid (ATRA), which is a retinoic acid receptor (RAR)α, RARß and RARγ agonist, on the differentiation from the hiPSC-derived foregut. Finally, to identify which RAR subtype was involved in the differentiation, we used synthetic agonists and antagonists of RARα and RARγ, which are known to be expressed in esophagus. RESULTS: We successfully generated stratified layers of cells expressing EEC marker genes that were positive for lugol staining. The enhancing effect of ATRA on EEC differentiation was clearly demonstrated with quantitative reverse transcription polymerase chain reaction, immunohistology, lugol-staining and RNA sequencing analyses. RARγ agonist and antagonist enhanced and suppressed EEC differentiation, respectively. RARα agonist had no effect on the differentiation. CONCLUSION: We revealed that RARγ activation promotes the differentiation of hiPSCs-derived foregut into EECs.

Directed differentiation of human induced pluripotent stem cells into mature stratified bladder urothelium.

For augmentation or reconstruction of urinary bladder after cystectomy, bladder urothelium derived from human induced pluripotent stem cells (hiPSCs) has recently received focus. However, previous studies have only shown the emergence of cells expressing some urothelial markers among derivatives of hiPSCs, and no report has demonstrated the stratified structure, which is a particularly important attribute of the barrier function of mature bladder urothelium. In present study, we developed a method for the directed differentiation of hiPSCs into mature stratified bladder urothelium. The caudal hindgut, from which the bladder urothelium develops, was predominantly induced via the high-dose administration of CHIR99021 during definitive endoderm induction, and this treatment subsequently increased the expressions of uroplakins. Terminal differentiation, characterized by the increased expression of uroplakins, CK13, and CK20, was induced with the combination of Troglitazone + PD153035. FGF10 enhanced the expression of uroplakins and the stratification of the epithelium, and the transwell culture system further enhanced such stratification. Furthermore, the barrier function of our urothelium was demonstrated by a permeability assay using FITC-dextran. According to an immunohistological analysis, the stratified uroplakin II-positive epithelium was observed in the transwells. This method might be useful in the field of regenerative medicine of the bladder.

Induced pluripotent stem cell-derived melanocyte precursor cells undergoing differentiation into melanocytes.

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self-renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte-specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high-level expression of WNT5A, ROR2, which are non-canonical WNT pathway markers, and its related receptor TGFßR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3ß inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non-canonical WNT signaling pathway.

The Generation of Human γδT Cell-Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture.

γδT cells constitute a small proportion of lymphocytes in peripheral blood. Unlike αßT cells, the anti-tumor activities are exerted through several different pathways in a MHC-unrestricted manner. Thus, immunotherapy using γδT cells is considered to be effective for various types of cancer. Occasionally, however, ex vivo expanded cells are not as effective as expected due to cell exhaustion. To overcome the issue of T-cell exhaustion, researchers have generated induced pluripotent stem cells (iPSCs) that harbor the same T-cell receptor (TCR) genes as their original T-cells, which provide nearly limitless sources for antigen-specific cytotoxic T lymphocytes (CTLs). However, these technologies have focused on αßT cells and require a population of antigen-specific CTLs, which are purified by cell sorting with HLA-peptide multimer, as the origin of iPS cells. In the present study, we aimed to develop an efficient and convenient system for generating iPSCs that harbor rearrangements of the TCRG and TCRD gene regions (γδT-iPSCs) without cell-sorting. We stimulated human whole peripheral blood mononuclear cell (PBMC) culture using Interleukin-2 and Zoledronate to activate γδT cells. Gene transfer into those cells with the Sendai virus vector resulted in γδT cell-dominant expression of exogenous genes. The introduction of reprogramming factors into the stimulated PBMC culture allowed us to establish iPSC lines. Around 70% of the established lines carried rearrangements at the TCRG and TCRD gene locus. The γδT-iPSCs could differentiate into hematopoietic progenitors. Our technology will pave the way for new avenues toward novel immunotherapy that can be applied for various types of cancer. Stem Cells Translational Medicine 2018;7:34-44.

Interleukin-6 blockade attenuates lung cancer tissue construction integrated by cancer stem cells.

In the present study, we successfully generated lung cancer stem cell (CSC)-like cells by introducing a small set of transcription factors into a lung cancer cell line. In addition to properties that are conventionally referred to as CSC properties, the lung induced CSCs exhibited the ability to form lung cancer-like tissues in vitro with vascular cells and mesenchymal stem cells, which showed structures and immunohistological patterns that were similar to human lung cancer tissues. We named them "lung cancer organoids". We found that interleukin-6 (IL-6), which was expressed in the lung induced CSCs, facilitates the formation of lung cancer organoids via the conversion of mesenchymal stem cells into alpha-smooth muscle actin (αSMA)-positive cells. Interestingly, the combination of anti-IL-6 antibody and cisplatin could destroy the lung cancer organoids, while cisplatin alone could not. Furthermore, IL-6 mRNA-positive cancer cells were found in clinical lung cancer samples. These results suggest that IL-6 could be a novel therapeutic target in lung cancer.

The Tissue-Reconstructing Ability of Colon CSCs Is Enhanced by FK506 and Suppressed by GSK3 Inhibition.

Cancer stem cells (CSC) are capable of reconstructing cancer tissues, are involved in both recurrence and metastasis, and contribute to therapeutic resistance. Therefore, elucidating the molecular mechanism in CSCs is important to successfully treat unresectable cancers. Previously, we observed that colon cancer stem-like cells can be induced from human colon cancer cell lines by retrovirally introducing OCT3/4, SOX2, and KLF4, and we have designated such cells as induced cancer stem cells (iCSC). In the current study, we used iCSCs to evaluate the molecular mechanism of colon CSCs and developed new methods to control them. The spheres that were derived in vitro from the iCSCs, but not those from parental cells, mimicked human colon cancer tissues in terms of their immunohistologic patterns; therefore, sphere-forming ability was assessed as a measure of the tissue-reconstructing ability of iCSCs. Interestingly, the calcineurin inhibitor FK506 enhanced the sphere-forming ability of iCSCs, whereas GSK3 inhibition by RNAi, CHIR99021, and valproic acid (VPA) impeded the sphere-forming ability and expansion of iCSCs. FK506 and GSK3 inhibition showed the opposite effect regarding the NFATc3 localization of iCSCs. These data reveal the crucial role that NFAT localization, as regulated by calcineurin and GSK3, plays in the tissue-reconstructing ability of colon cancer stem cells and the potential of GSK3 inhibitors, such as VPA, in colon cancer stem cell-targeting therapy.Implications: This study identifies signaling pathways that contribute to the tissue-reconstructing capacity of colon CSCs and suggests that clinically used drugs could be repurposed to improve unresectable colon cancers. Mol Cancer Res; 15(10); 1455-66. ©2017 AACR.

Epigenetic Variation between Human Induced Pluripotent Stem Cell Lines Is an Indicator of Differentiation Capacity.

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells.

We examined the gene expression and DNA methylation of 49 human induced pluripotent stem cells (hiPSCs) and 10 human embryonic stem cells and found overlapped variations in gene expression and DNA methylation in the two types of human pluripotent stem cell lines. Comparisons of the in vitro neural differentiation of 40 hiPSCs and 10 human embryonic stem cells showed that seven hiPSC clones retained a significant number of undifferentiated cells even after neural differentiation culture and formed teratoma when transplanted into mouse brains. These differentiation-defective hiPSC clones were marked by higher expression levels of several genes, including those expressed from long terminal repeats of specific human endogenous retroviruses. These data demonstrated a subset of hiPSC lines that have aberrant gene expression and defective potential in neural differentiation, which need to be identified and eliminated before applications in regenerative medicine.

Tsix RNA and the germline factor, PRDM14, link X reactivation and stem cell reprogramming.

Transitions between pluripotent and differentiated states are marked by dramatic epigenetic changes. Cellular differentiation is tightly linked to X chromosome inactivation (XCI), whereas reprogramming to induced pluripotent stem cells (iPSCs) is associated with X chromosome reactivation (XCR). XCR reverses the silent state of the inactive X, occurring in mouse blastocysts and germ cells. In spite of its importance, little is known about underlying mechanisms. Here, we examine the role of the long noncoding Tsix RNA and the germline factor, PRDM14. In blastocysts, XCR is perturbed by mutation of either Tsix or Prdm14. In iPSCs, XCR is disrupted only by PRDM14 deficiency, which also affects iPSC derivation and maintenance. We show that Tsix and PRDM14 directly link XCR to pluripotency: first, PRDM14 represses Rnf12 by recruiting polycomb repressive complex 2; second, Tsix enables PRDM14 to bind Xist. Thus, our study provides functional and mechanistic links between cellular and X chromosome reprogramming.