Biallelic CLCN2 mutations cause retinal degeneration by impairing retinal pigment epithelium phagocytosis and chloride channel function.

CLCN2 encodes a two-pore homodimeric chloride channel protein (CLC-2) that is widely expressed in human tissues. The association between Clcn2 and the retina is well-established in mice, as loss-of-function of CLC-2 can cause retinopathy in mice; however, the ocular phenotypes caused by CLCN2 mutations in humans and the underlying mechanisms remain unclear. The present study aimed to define the ocular features and reveal the pathogenic mechanisms of CLCN2 variants associated with retinal degeneration in humans using an in vitro overexpression system, as well as patient-induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) cells and retinal organoids (ROs). A patient carrying the homozygous c.2257C > T (p.R753X) nonsense CLCN2 mutation was followed up for > 6 years. Ocular features were comprehensively characterized with multimodality imaging and functional examination. The patient presented with severe bilateral retinal degeneration with loss of photoreceptor and RPE. In vitro, mutant CLC-2 maintained the correct subcellular localization, but with reduced channel function compared to wild-type CLC-2 in HEK293T cells. Additionally, patient iPSC-derived RPE cells carrying the CLCN2 mutation exhibited dysfunctional ClC-2 chloride channels and outer segment phagocytosis. Notably, these functions were rescued following the repair of the CLCN2 mutation using the CRISPR-Cas9 system. However, this variant did not cause significant photoreceptor degeneration in patient-derived ROs, indicating that dysfunctional RPE is likely the primary cause of biallelic CLCN2 variant-mediated retinopathy. This study is the first to establish the confirmatory ocular features of human CLCN2-related retinal degeneration, and reveal a pathogenic mechanism associated with biallelic CLCN2 variants, providing new insights into the cause of inherited retinal dystrophies.

Development and validation of a machine learning model to predict prognosis in HIV-negative cryptococcal meningitis patients: a multicenter study.

PURPOSE: To predict prognosis in HIV-negative cryptococcal meningitis (CM) patients by developing and validating a machine learning (ML) model. METHODS: This study involved 523 HIV-negative CM patients diagnosed between January 1, 1998, and August 31, 2022, by neurologists from 3 tertiary Chinese centers. Prognosis was evaluated at 10 weeks after the initiation of antifungal therapy. RESULTS: The final prediction model for HIV-negative CM patients comprised 8 variables: Cerebrospinal fluid (CSF) cryptococcal count, CSF white blood cell (WBC), altered mental status, hearing impairment, CSF chloride levels, CSF opening pressure (OP), aspartate aminotransferase levels at admission, and decreased rate of CSF cryptococcal count within 2 weeks after admission. The areas under the curve (AUCs) in the internal, temporal, and external validation sets were 0.87 (95% CI 0.794-0.944), 0.92 (95% CI 0.795-1.000), and 0.86 (95% CI 0.744-0.975), respectively. An artificial intelligence (AI) model was trained to detect and count cryptococci, and the mean average precision (mAP) was 0.993. CONCLUSION: A ML model for predicting prognosis in HIV-negative CM patients was built and validated, and the model might provide a reference for personalized treatment of HIV-negative CM patients. The change in the CSF cryptococcal count in the early phase of HIV-negative CM treatment can reflect the prognosis of the disease. In addition, utilizing AI to detect and count CSF cryptococci in HIV-negative CM patients can eliminate the interference of human factors in detecting cryptococci in CSF samples and reduce the workload of the examiner.

Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality.

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.

An Optimized System for Effective Derivation of Three-Dimensional Retinal Tissue via Wnt Signaling Regulation.

Effective derivation of three-dimensional (3D) retinal tissue from human-induced pluripotent stem cells (hiPSCs) could provide models for drug screening and facilitate patient-specific retinal cell replacement therapy. However, some hiPSC lines cannot undergo 3D self-organization and show inadequate differentiation efficiency to meet clinical demand. In this study, we developed an optimized system for derivation of 3D retinal tissue. We found that the Wnt signaling pathway antagonist Dickkopf-related protein 1 (DKK-1) rescued the inability of differentiated retinal progenitors to self-organize. By evaluating DKK-1 expression and supplying DKK-1 if necessary, retinal organoids were differentiated from six hiPSC lines, which were reprogramed from three common initiating cell types. Retinal tissues derived from the optimized system were well organized and capable of surviving for further maturation. Thus, using this system, we generated retinal tissues from various hiPSC lines with high efficiency. This novel system has many potential applications in regenerative therapy and precision medicine. Stem Cells 2018;36:1709-1722.

A novel mutation of WFS1 gene in a Chinese patient with Wolfram syndrome: a case report.

BACKGROUND: Wolfram syndrome (WS), caused by mutations of the Wolfram syndrome 1 (WFS1) gene on chromosome 4p16.1, is an autosomal recessive disorder characterized by diabetes insipidus (DI), neuro-psychiatric disorders, hearing deficit, and urinary tract anomalies. CASE PRESENTATION: Here we report a 11-year-old Chinese boy who presented with visual loss, was suspected with optic neuritis (ON) or neuromyelitis optica (NMO) and referred to our department for further diagnosis. Finally he was diagnosed with WS because of diabetes mellitus (DM) and optic atrophy (OA). Eight exons and flanking introns of WFS1 gene were analyzed by sequencing. A novel mutation c.1760G > A in WFS1 gene of exon 8 was identified. CONCLUSION: This report reviews a case of WS associated with a novel mutation, c.1760G > A in WFS1 gene of exon 8, and emphasizes that WS should be taken into account for juveniles with visual loss and diabetes mellitus.

Generation of two hiPSC lines carrying compound heterozygous RDH12 mutations in a LCA patient.

Leber's congenital amaurosis (LCA) is a complex inherited retinal dystrophy characterized by severe vision loss and even blindness early in life, caused by more than 38 genes. Variations in RDH12 were found to be responsible for LCA. We successfully generated two induced pluripotent stem cell lines from a patient diagnosed with LCA carrying the RDH12 compound heterozygous mutations c.524C>T (p.Ser175Leu) and c.806C>G (p.Ala269Gly). Both iPSC lines displayed differentiation potential in vitro, exhibited normal karyotype and expressed pluripotency markers. These iPSC lines will act as a tool for studying the pathogenesis and treatment of RDH12-related LCA.

Distinct serum apolipoprotein A-I levels in neuromyelitis optica and acute transverse myelitis.

OBJECTIVE: NMO and ATM are intertwined both clinically and pathologically. Apolipoprotein (apo) A-I, the main apolipoprotein of HDL, plays an important role in lipid metabolism in the cerebrospinal fluid and is known to suppress pro-inflammatory cytokines generated by activated T cells in some autoimmune diseases as an immune regulator. However, the differences in the levels of serum apoA-I between NMO and ATM patients are unclear. METHODS: In the present study, serum apo A-I levels were measured in 53 NMO patients, 45 ATM patients and 49 healthy subjects. We tested serum apoA-I levels in all participants and investigated EDSS scores of patients with NMO and ATM. Statistical analyses were performed by using SPSS statistical software. RESULT: We found that serum apoA-I levels in patients with NMO were significantly lower in comparison to those with ATM. We also found that serum levels of apoA-I was lower in male subjects in comparison to the female subjects in all groups although these differences were not statistically significant in patients with NMO or ATM. It is also shown in our study that serum apoA-I levels in patients with NMO were significantly elevated after receiving a high dosage of intravenous corticosteroids over a period of one week. However, we did not find any correlation between the apoA-I levels and disease disability. CONCLUSION: From this study, we concluded that serum levels of apoA-I were lower in NMO patients compared to patients with ATM. Serum apoA-I studies might provide some useful clues to differentiate NMO cases from ATM cases.

Generation and characterization of two induced pluripotent stem cell lines from conjunctiva of a retinoblastoma patient.

Retinoblastoma (RB) is a common intraocular malignancy mostly caused by variation of the tumour suppressor gene RB1. In this study, we successfully generated two induced pluripotent stem cell (iPSC) lines from an infant with non-heritable RB. Both cell clones exhibited typical iPSC characteristics with normal karyotypes, consistent pluripotency markers expression and the capability of trilineage differentiation.

Generation and characterization of two iPSC lines carrying heterozygous or homozygous nonsense mutation in PROM1 gene from a single family.

PROM1-related retinal dystrophy (PROM1-RD) is a group of hereditary retinal disorder characterized by the progressive damage of the photoreceptors. We generated and identified two induced pluripotent stem cell (iPSC) lines carrying homozygous or heterozygous nonsense mutation c.619G > T (p.E207X) in PROM1 gene from a patient with PROM1-RD and his healthy mother, respectively. Both iPSC lines maintained the typical stem cell morphology, genomic stability and pluripotency. These iPSC lines have great potential to elucidate the disease mechanisms and develop the feasible treatments of PROM1-RD.

Generation of an RCVRN-eGFP Reporter hiPSC Line by CRISPR/Cas9 to Monitor Photoreceptor Cell Development and Facilitate the Cell Enrichment for Transplantation.

Stem cell-based cell therapies are considered to be promising treatments for retinal disorders with dysfunction or death of photoreceptors. However, the enrichment of human photoreceptors suitable for transplantation has been highly challenging so far. This study aimed to generate a photoreceptor-specific reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 genome editing, which harbored an enhanced green fluorescent protein (eGFP) sequence at the endogenous locus of the pan photoreceptor marker recoverin (RCVRN). After confirmation of successful targeting and gene stability, three-dimensional retinal organoids were induced from this reporter line. The RCVRN-eGFP reporter faithfully replicated endogenous protein expression of recoverin and revealed the developmental characteristics of photoreceptors during retinal differentiation. The RCVRN-eGFP specifically and steadily labeled photoreceptor cells from photoreceptor precursors to mature rods and cones. Additionally, abundant eGFP-positive photoreceptors were enriched by fluorescence-activated cell sorting, and their transcriptome signatures were revealed by RNA sequencing and data analysis. Moreover, potential clusters of differentiation (CD) biomarkers were extracted for the enrichment of photoreceptors for clinical applications, such as CD133 for the positive selection of photoreceptors. Altogether, the RCVRN-eGFP reporter hiPSC line was successfully established and the first global expression database of recoverin-positive photoreceptors was constructed. These achievements will provide a powerful tool for dynamically monitoring photoreceptor cell development and purification of human photoreceptors, thus facilitating photoreceptor cell therapy for advanced retinal disorders.

Spatial and Temporal Development of Müller Glial Cells in hiPSC-Derived Retinal Organoids Facilitates the Cell Enrichment and Transcriptome Analysis.

Müller glial cells (MGCs) play important roles in human retina during physiological and pathological conditions. However, the development process of human MGCs in vivo remains unclear, and how to obtain large numbers of human MGCs with high quality faces technical challenges, which hinder the further study and application of MGCs. Human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) with all retinal cell subtypes provide an unlimited cell resource and a platform for the studies of retinal development and disorders. This study explored the development of human MGCs in hiPSC-derived ROs and developed an approach to select and expand the induced MGCs (iMGCs). In ROs, retinal progenitor cells progressively differentiated into SOX9+ Ki67- MGC precursors during differentiation day (D) 60 to D90, while mature MGCs expressing markers CRALBP and GS gradually appeared since D120, which spanned the entire thickness of the neural retina layer. Cells isolated from ROs aged older than 120 days was an optimal source for the enrichment of iMGCs with high purity and expansion ability. They had typical features of human MGCs in morphological, structural, molecular and functional aspects, and could be passaged serially at least 10 times, yielding large numbers of cells in a short period. The transcriptome pattern of the expanded iMGCs was also revealed. This study firstly clarified the timecourse of human MGC development in the RO model, where the iMGCs could be enriched and expanded, paving the way for downstream investigation and application in MGC-related retinal disorders.

Amniotic Membrane Enhances the Characteristics and Function of Stem Cell-Derived Retinal Pigment Epithelium Sheets by Inhibiting the Epithelial-Mesenchymal Transition.

Human pluripotent stem cell-derived retinal pigment epithelium (iRPE) is an attractive cell source for disease modeling and cell replacement therapy of retinal disorders with RPE defects. However, there are still challenges to develop appropriate culture conditions close to in vivo microenvironment to generate iRPE sheets, which mimic more faithfully the characteristics and functions of the human RPE cells. Here, we developed a simple, novel platform to construct authentic iRPE sheets using human amniotic membrane (hAM) as a natural scaffold. The decellularized hAM (dAM) provided a Bruch's membrane (BM)-like bioscaffold, supported the iRPE growth and enhanced the epithelial features, polarity distribution and functional features of iRPE cells. Importantly, RNA-seq analysis was performed to compare the transcriptomes of iRPE cells cultured on different substrates, which revealed the potential mechanism that dAM supported and promoted iRPE growth was the inhibition of epithelial-mesenchymal transition (EMT). The tissue-engineered iRPE sheets survived and kept monolayer when transplanted into the subretinal space of rabbits. All together, our results indicate that the dAM imitating the natural BM allows for engineering authentic human RPE sheets, which will provide valuable biomaterials for disease modeling, drug screening and cell replacement therapy of retinal degenerative diseases. STATEMENT OF SIGNIFICANCE: Engineered RPE sheets have a great advantage over RPE cell suspension for transplantation as they support RPE growth in an intact monolayer which RPE functions are dependent on. The substrates for RPE culture play a critical role to maintain the physiological functions of the RPE in stem cell therapies for patients with retinal degeneration. In this study, we constructed engineered iRPE sheets on the decellularized human amniotic membrane scaffolds, which contributed to enhancing epithelial features, polarity distribution and functional features of iRPE. dAM exhibited the ability of anti-epithelial mesenchymal transition to support iRPE growth. Furthermore, the results of transplantation in vivo demonstrated the feasibility of iRPE sheets in retina regenerative therapy. Engineering RPE sheets on dAM is a promising strategy to facilitate the development of iRPE replacement therapy and retinal disease modeling.

Generation of a X-linked juvenile retinoschisis patient-derived induced pluripotent stem cell line ZOCi004-A.

X-linked juvenile retinoschisis (XLRS), caused by the mutation of RS1 gene, is one of the most common causes of macular degeneration for male adolescents. The mutations and clinical manifestations of the disease are diverse. Neither the relationship between the genotypes and phenotypes, nor the radical treatment like gene therapy has been found by now. Retrospective studies have shown that carbonic anhydrase inhibitors can help reduce cysts. However, the specifically pharmacological mechanism remains unknown. Here, we culture induced pluripotent stem cells by drawing peripheral blood from a patient with XLRS, which are supposed to facilitate related researches.

Vascular endothelial growth factor-B gene transfer exacerbates retinal and choroidal neovascularization and vasopermeability without promoting inflammation.

PURPOSE: The role of vascular endothelial growth factor (VEGF)-B in the eye is poorly understood. The present study was conducted to evaluate the effect of overexpression of VEGF-B via adeno-associated virus (AAV) gene transfer on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB). METHODS: Three recombinant AAV vectors were prepared, expressing the 167 (AAV-VEGF-B167) or 186 amino acid isoform (AAV-VEGF-B186) of VEGF-B or the green fluorescent protein (GFP) reporter gene (AAV-GFP). Approximately 1 x 109 viral genome copies of AAV-VEGF-B167, AAV-VEGF-B186, or AAV-GFP were intraocularly injected. The efficacy of the gene transfer was assessed by directly observing GFP, by immunohistochemistry, or by real-time PCR. A leukostasis assay using fluorescein isothiocyanate-conjugated Concanavalin A was used to evaluate inflammation. The BRB was assessed using a quantitative assay with ³H-mannitol as a tracer. Retinal neovascularization (NV) was assessed at postnatal day 17 in oxygen-induced ischemic retinopathy after intravitreal injection of AAV-VEGF-B in left eyes and AAV-GFP in right eyes at postnatal day 7. Two weeks after injection of AAV vectors, choroidal NV was generated by laser photocoagulation and assessed 2 weeks later. RESULTS: GFP expression was clearly demonstrated, primarily in the retinal pigment epithelium (RPE) and outer retina, 1-6 weeks after delivery. mRNA expression levels of VEGF-B167 and VEGF-B186 were 5.8 and 12 fold higher in the AAV-VEGF-B167- and AAV-VEGF-B186-treated groups, respectively. There was no evidence of an inflammatory response or vessel abnormality following injection of the vectors in normal mice; however, VEGF-B increased retinal and choroidal neovascularization. AAV-VEGF-B186, but not AAV-VEGF-B167, enhanced retinal vascular permeability. CONCLUSIONS: VEGF-B overexpression promoted pathological retinal and choroidal NV and BRB breakdown without causing inflammation, which is associated with the progression of diabetic retinopathy and age-related macular degeneration, showing that these complications are not dependent on inflammation. VEGF-B targeting could benefit antiangiogenic therapy.

E13.5 retinal progenitors induce mouse bone marrow mesenchymal stromal cells to differentiate into retinal progenitor-like cells.

BACKGROUND AIMS: Retinal progenitor cells (RPC) are an excellent resource for retinal replacement therapy but usually unavailable. We attempted to induce bone marrow mesenchymal stromal cells (BMSC) into RPC. METHODS: BMSC and embryonic day 13.5 (E13.5) RPC derived from wild-type or enhanced green fluorescence protein (EGFP) transgenic (Egfp(+/+)) mice were co-cultured in a transwell or re-aggregation system. Gene and protein expressions were investigated by reverse transcription-polymerase chain reaction (PCR) and immunofluorescence, respectively. Spontaneous cell fusion was evaluated by Chloromethylbenzamido derivative of 1,1'- dioctadecyl-3,3,3',3' - tetramethylindocarbocyanine perchlorate (CM-DiI) labeling together with EGFP tracing. RESULTS: BMSC from both wild-type and Egfp(+/+) mice displayed similar spindle shapes. The undifferentiated BMSC already expressed immature neural markers but did not express Nfl, Gfap or the retina-related genes Pax6, Math5 and Brn3b. When co-cultured with E13.5 RPC in the transwell system, BMSC displayed transient expression of early retinal development genes, including Pax6, Math5 and Brn3b at 3 days, as well as long-term expression of Nfl (up to 21 days). No expression of the late photoreceptor gene rhodopsin could be detected at any time. In re-aggregation co-culture, E13.5 RPC induced EGFP-positive BMSC to express not only the early retinal development genes but also the late gene rhodopsin. Furthermore, a small fraction of BMSC could be induced to express the synaptophysin protein. Re-aggregation co-culture of CM-DiI-labeled BMSC and EGFP-positive E13.5 RPC displayed minimal co-localization of the two fluorescence signals. CONCLUSIONS: E13.5 RPC are capable of inducing BMSC towards an RPC fate. The differentiation is independent of cell fusion. Cytokines and cell-cell interactions exert this induction effect, but they have different functions.

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells.

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Human retinal organoids release extracellular vesicles that regulate gene expression in target human retinal progenitor cells.

The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with Gene Ontology (GO) pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.

Establishment of a Rapid Lesion-Controllable Retinal Degeneration Monkey Model for Preclinical Stem Cell Therapy.

BACKGROUND: Retinal degenerative disorders (RDs) are the main cause of blindness without curable treatment. Our previous studies have demonstrated that human-induced pluripotent stem cells can differentiate into retinal organoids with all subtypes of retina, which provides huge promise for treating these diseases. Before these methods can be realized, RD animal models are required to evaluate the safety and efficacy of stem cell therapy and to develop the surgical tools and procedures for cell transplantation in patients. This study involved the development of a monkey model of RD with controllable lesion sites, which can be rapidly prepared for the study of preclinical stem cell therapy among other applications. METHODS: Sodium nitroprusside (SNP) in three doses was delivered into the monkey eye by subretinal injection (SI), and normal saline was applied as control. Structural and functional changes of the retinas were evaluated via multimodal imaging techniques and multifocal electroretinography (mfERG) before and after the treatment. Histological examination was performed to identify the target layer of the affected retina. The health status of monkeys was monitored during the experiment. RESULTS: Well-defined lesions with various degrees of retinal degeneration were induced at the posterior pole of retina as early as 7 days after SNP SI. The damage of SNP was dose dependent. In general, 0.05 mM SNP caused mild structural changes in the retina; 0.1 mM SNP led to the loss of outer retinal layers, including the outer plexiform layer (OPL), outer nuclear layer (ONL), and retinal pigment epithelium (RPE); while 0.2 mM SNP impacted the entire layer of the retina and choroid. MfERG showed reduced amplitude in the damaged region. The structural and functional damages were not recovered at 7-month follow-up. CONCLUSION: A rapidly induced lesion site-controllable retinal degeneration monkey model was established by the subretinal administration of SNP, of which the optimal dose is 0.1 mM. This monkey model mimics the histological changes of advanced RDs and provides a valuable platform for preclinical assessment of stem cell therapy for RDs.

Generation of an iPSC line (SKLOi001-A) from a patient with CLCN2-related leukoencephalopathy.

CLCN2-related leukoencephalopathy (CC2L) is a rare disease due to autosomal recessive loss-of-function mutations in CLCN2 gene. We generated an induced pluripotent stem cell (iPSC) line (SKLOi001-A) from urine cells isolated from a CC2L patient carrying a homozygotic mutation: c.2257C>T (p.Arg753*) in CLCN2 gene via an integration-free methods. The established iPSC line kept the CLCN2 mutation and displayed a normal karyotype, expressed pluripotency markers, showed differentiation potential. This newly iPSC line could be served as a possible tool to unravel the mechanisms underlying CLCN2-associated diseases and screen drugs for the treatment.

Role of MEF feeder cells in direct reprogramming of mousetail-tip fibroblasts.

Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c-Myc and Klf4 when co-cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder-free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine.