Glycine decarboxylase regulates the maintenance and induction of pluripotency via metabolic control.

Reprogramming of 'adult' differentiated somatic cells to 'embryonic' pluripotent stem cells accompanied by increased rate of glycolysis. Conversely, glycolysis triggers accumulation of advanced glycation end products (AGEs), a potential causative factor in aging, by promoting methylglyoxal production. Therefore, it is reasonable that pluripotent stem cells (PSCs) would specifically regulate glycolysis to maintain their embryonic features. In this study, we focused on glycine decarboxylase (GLDC), a key enzyme in the glycine cleavage system that regulates glycolysis and methylglyoxal production in cancer. GLDC was exclusively expressed in PSCs, and inhibition of this enzyme induced alterations of metabolome and AGE accumulation, thereby suppressing the embryonic pluripotent state. Surprisingly, the level of accumulated AGEs in somatic cells gradually decreased during reprogramming, ultimately disappearing in iPSCs. In addition, ectopic expression of GLDC or treatment with the AGE inhibitor LR-90 promoted reprogramming. Together, these findings suggest that GLDC-mediated regulation of glycolysis and controlling AGE accumulation is related to maintenance and induction of pluripotency.

Inhibitory effect of celastrol on adipogenic differentiation of human adipose-derived stem cells.

Control of adipogenesis in mesenchymal stem cells (MSCs) offers enormous potential for management of obesity- and aging-related diseases. Celastrol, the traditional Chinese medicine extracted from Tripterygium wilfordi, exhibits anti-obesity effects in in vitro and in vivo murine models. This study describes how celastrol affects multilineage differentiation potential of human adipose-derived stem cells (hADSCs). We performed in vitro adipogenic differentiation of hADSCs and investigated how celastrol-induced lipid accumulation and expression of adipocyte differentiation markers varied with dose, duration, and donor age. In addition, we assessed the effect of celastrol on osteogenic and chondrogenic differentiation of hADSCs. During adipogenic induction of hADSCs, the inhibitory effect of celastrol on lipid accumulation and adipogenesis depended on dose, duration, time of administration, and individual donor. Inhibition was mediated by proliferator-activated receptor-γ (PPARG) and CCAAT/enhancer-binding protein alpha (CEBPA). Celastrol also suppressed differentiation of hADSCs into the osteogenic and chondrogenic lineages. Celastrol plays a regulatory role in multilineage differentiation of human MSCs. Our findings provide important insights regarding management of obesity and stem cell therapy.

Utilizing stem cell-secreted molecules as a versatile toolbox for skin regenerative medicine.

Stem cells are recognized as an important target and tool in regenerative engineering. In this study, we explored the feasibility of engineering amniotic fluid-derived mesenchymal stem cell-secreted molecules (afMSC-SMs) as a versatile bioactive material for skin regenerative medicine applications in a time- and cost-efficient and straightforward manner. afMSC-SMs, obtained in powder form through ethanol precipitation, effectively contributed to preserving the self-renewal capacity and differentiation potential of primary human keratinocytes (pKCs) in a xeno-free environment, offering a potential alternative to traditional culture methods for their long-term in vitro expansion, and allowed them to reconstitute a fully stratified epithelium sheet on human dermal fibroblasts. Furthermore, we demonstrated the flexibility of afMSC-SMs in wound healing and hair regrowth through injectable hydrogel and nanogel-mediated transdermal delivery systems, respectively, expanding the pool of regenerative applications. This cell-free approach may offer several potential advantages, including streamlined manufacturing processes, scalability, controlled formulation, longer shelf lives, and mitigation of risks associated with living cell transplantation. Accordingly, afMSC-SMs could serve as a promising therapeutic toolbox for advancing cell-free regenerative medicine, simplifying their broad applicability in various clinical settings.

Establishment of Immortalized Human Endometriotic Stromal Cell Line from Ectopic Lesion of a Patient with Endometriosis.

Endometriosis is an estrogen-dependent inflammatory disease characterized by the growth of endometrial-like tissues containing endometrial stromal cells and glandular epithelium outside the uterine cavity. An insufficient response to progesterone contributes to disease progression and systemic inflammation during the pathogenesis of endometriosis. Patients with endometriosis usually experience painful symptoms, dysmenorrhea, and infertility, which contribute to a significant reduction in their quality of life. To determine the possible molecular mechanisms of endometriosis and explore novel therapeutic targets, we derived primary human ovarian endometriotic stromal cells (hOESCs) from a patient of reproductive age with ovarian endometriosis. In this study, we successfully established immortalized human ovarian endometriotic stromal cell lines (ihOESCs) using primary stromal cells obtained from endometriotic lesions to overcome short lifespan and growth inhibition. Immortalization of hOESCs with human telomerase reverse transcriptase (hTERT) transfection led to cells that maintained a proliferative state under passage culture conditions without mutagenesis during cellular senescence. The morphology and karyotype of ihOESCs were unchanged compared with those of hOESCs. Moreover, ihOESCs were continuously positive for vimentin and negative for E-cadherin expression. Following decidual stimuli and inflammatory responses, both hOESCs and ihOESCs sensitively express decidualization markers and proinflammatory cytokines. Collectively, we characterized ihOESCs to maintain their phenotypic and functional properties with a longer lifespan and normal physiological responses than those of hOESCs. These immortalized cells could aid in a detailed understanding of the pathological mechanisms of endometriosis.

Human induced neural stem cells support functional recovery in spinal cord injury models.

Spinal cord injury (SCI) is a clinical condition that leads to permanent and/or progressive disabilities of sensory, motor, and autonomic functions. Unfortunately, no medical standard of care for SCI exists to reverse the damage. Here, we assessed the effects of induced neural stem cells (iNSCs) directly converted from human urine cells (UCs) in SCI rat models. We successfully generated iNSCs from human UCs, commercial fibroblasts, and patient-derived fibroblasts. These iNSCs expressed various neural stem cell markers and differentiated into diverse neuronal and glial cell types. When transplanted into injured spinal cords, UC-derived iNSCs survived, engrafted, and expressed neuronal and glial markers. Large numbers of axons extended from grafts over long distances, leading to connections between host and graft neurons at 8 weeks post-transplantation with significant improvement of locomotor function. This study suggests that iNSCs have biomedical applications for disease modeling and constitute an alternative transplantation strategy as a personalized cell source for neural regeneration in several spinal cord diseases.

Self-replicative mRNA-mediated generation of induced pluripotent stem cell line from a 1-year-old Leigh syndrome patient with mitochondrial DNA cytochrome b mutation.

Leigh syndrome is a progressive neurodegenerative disease due to defects in the mitochondrial genes, including mitochondrial DNA cytochrome b (MTCYB) mutation, that typically begins in infancy or early childhood. Exercise intolerance and fatigue are common symptoms of mitochondrial disorders. Here, we generated induced pluripotent stem cell (iPSC) line from a 1-year-old patient with Leigh syndrome with MTCYB through temporal expression of exogenes, synthetic self-replicative mRNAs which were regulated by B18R protein. The established iPSCs showed expression of various pluripotency markers, a normal karyotype and differentiation potential to three germ layers in vitro while retaining MTCYB mutation.

Generation of a WA14 hESC sub-line carrying a hemizygous ABCD1 (C.1696_1710 del) mutation introduced by CRISPR/Cas9 technology.

ATP-binding cassette transporter subfamily D member 1 (ABCD1) gene is a member of ABC transporter super family, which conduct peroxisomal import of very long chain fatty acid and crucial underlying factor that induces X-linked adrenoleukodystrophy (X-ALD) when the gene is defected. Here, we report the generation of a human embryonic stem cell sub-line harboring a hemizygous ABCD1 mutation (C.1696_1710 del) using CRISPR/Cas9 system. Established line expresses pluripotency marker genes, can be differentiated to three germ layers, and maintains a normal karyotype.

Isolation of mesenchymal stem cells from Pap smear samples.

OBJECTIVE: Exploiting their ability to differentiate into mesenchymal lineages like cartilage, bone, fat, and muscle, and to elicit paracrine effects, mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders. One of accessible sources of MSC is the samples used for Papanicolaou (Pap) test, which is a cervical screening method for detecting potentially pre-cancerous and cancerous alterations in the cervical cells and to diagnose genetic abnormalities in fetuses. This study aimed to identify and isolate the stem cells from Pap smear samples collected from pregnant women, and to trace the origin of these cells to maternal or fetal tissue, and characterize their stem cell properties. METHODS: To investigate the possibility and efficiency of establishing MSC lines from the Pap smear samples, we were able to establish 6 cell lines from Pap smear samples from 60 pregnant women at different stages of gestation. RESULTS: The 3 cell lines randomly selected among the 6 established in this study, displayed high proliferation rates, several characteristics of MSCs, and the capacity to differentiate into adipocytes, osteocytes, and chondrocytes. Our study identified that the stem cell lines obtainable from Pap smear sampling were uterine cervical stromal cells (UCSCs) and had 10% efficiency of establishment. CONCLUSION: Despite their low efficiency of establishment, human UCSCs from Pap smear samples can become a simple, safe, low-cost, and donor-specific source of MSCs for stem cell therapy and regenerative medicine.

Generation of an induced pluripotent stem cell (iPSC) line from a 42-year-old adult cerebral type X-linked adrenoleukodystrophy (X-ALD) patient.

X-linked Adrenoleukodystrophy (X-ALD) is a neuro-metabolic disorder that is caused by malfunction of a peroxisomal transporter protein, adenosine ATP-binding cassette transporter superfamily D member 1 (ABCD1). We established an induced pluripotent stem cell (iPSC) line from a 42-year-old male X-ALD patient-derived dermal fibroblasts with Sendai virus-mediated reprogramming. Established iPSCs stably expanded, expressed genes of pluripotency, and maintained normal karyotype. In vitro differentiation assay revealed the characteristics of all three germ layers.

Generation of Anterior Hindbrain-Specific, Glial-Restricted Progenitor-Like Cells from Human Pluripotent Stem Cells.

Engraftment of oligodendrocyte progenitor cells (OPCs), which form myelinating oligodendrocytes, has the potential to treat demyelinating diseases such as multiple sclerosis. However, conventional strategies for generating oligodendrocytes have mainly focused on direct differentiation into forebrain- or spinal cord-restricted oligodendrocytes without establishing or amplifying stem/progenitor cells. Taking advantage of a recently established culture system, we generated expandable EN1- and GBX2-positive glial-restricted progenitor-like cells (GPLCs) near the anterior hindbrain. These cells expressed PDGFRα, CD9, S100ß, and SOX10 and mostly differentiated into GFAP-positive astrocytes and MBP-positive oligodendrocytes. RNA-seq analysis revealed that the transcriptome of GPLCs was similar to that of O4-positive OPCs, but distinct from that of rosette-type neural stem cells. Notably, engrafted GPLCs not only differentiated into GFAP-positive astrocytes but also myelinated the brains of adult shiverer mice 8 weeks after transplantation. Our strategy for establishing anterior hindbrain-specific GPLCs with gliogenic potency will facilitate their use in the treatment of demyelinating diseases and studies of the molecular mechanisms underlying glial development in the hindbrain.

Overexpression of Nanog in amniotic fluid-derived mesenchymal stem cells accelerates dermal papilla cell activity and promotes hair follicle regeneration.

Alopecia, one of the most common chronic diseases, can seriously affect a patient's psychosocial life. Dermal papilla (DP) cells serve as essential signaling centers in the regulation of hair growth and regeneration and are associated with crosstalk between autocrine/paracrine factors and the surrounding environment. We previously demonstrated that amniotic fluid-derived mesenchymal stem cell-conditioned medium (AF-MSC-CM) accelerates hair regeneration and growth. The present study describes the effects of overexpression of a reprogramming factor, Nanog, on MSC properties, the paracrine effects on DP cells, and in vivo hair regrowth. First, we examined the in vitro proliferation and lifespan of AF-MSCs overexpressing reprogramming factors, including Oct4, Nanog, and Lin28, alone or in combination. Among these factors, Nanog was identified as a key factor in maintaining the self-renewal capability of AF-MSCs by delaying cellular senescence, increasing the endogenous expression of Oct4 and Sox2, and preserving stemness. Next, we evaluated the paracrine effects of AF-MSCs overexpressing Nanog (AF-N-MSCs) by monitoring secretory molecules related to hair regeneration and growth (IGF, PDGF, bFGF, and Wnt7a) and proliferation of DP cells. In vivo studies revealed that CM derived from AF-N-MSCs (AF-N-CM) accelerated the telogen-to-anagen transition in hair follicles (HFs) and increased HF density. The expression of DP and HF stem cell markers and genes related to hair induction were higher in AF-N-CM than in CM from AF-MSCs (AF-CM). This study suggests that the secretome from autologous MSCs overexpressing Nanog could be an excellent candidate as a powerful anagen inducer and hair growth stimulator for the treatment of alopecia.

mRNA-Driven Generation of Transgene-Free Neural Stem Cells from Human Urine-Derived Cells.

Human neural stem cells (NSCs) hold enormous promise for neurological disorders, typically requiring their expandable and differentiable properties for regeneration of damaged neural tissues. Despite the therapeutic potential of induced NSCs (iNSCs), a major challenge for clinical feasibility is the presence of integrated transgenes in the host genome, contributing to the risk for undesired genotoxicity and tumorigenesis. Here, we describe the advanced transgene-free generation of iNSCs from human urine-derived cells (HUCs) by combining a cocktail of defined small molecules with self-replicable mRNA delivery. The established iNSCs were completely transgene-free in their cytosol and genome and further resembled human embryonic stem cell-derived NSCs in the morphology, biological characteristics, global gene expression, and potential to differentiate into functional neurons, astrocytes, and oligodendrocytes. Moreover, iNSC colonies were observed within eight days under optimized conditions, and no teratomas formed in vivo, implying the absence of pluripotent cells. This study proposes an approach to generate transplantable iNSCs that can be broadly applied for neurological disorders in a safe, efficient, and patient-specific manner.

Generation of induced pluripotent stem cell (iPSC) line from a 36-year-old Charcot-Marie-Tooth disease patient with GJB1 mutation (CMTX).

Charcot-Marie-Tooth disease (CMTX) is inherited neurological disorder caused by gap junction beta 1 gene (GJB1) mutation. We generated induced pluripotent stem cell (iPSC) line from 36-year-old CMTX disease patient by electroporation of skin fibroblasts with episomal vectors encoding OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA-p53. Established iPSCs expressed various pluripotency markers, had differentiation potential of three germ layers in vitro, had normal karyotype and retained GJB1 mutation. This CMT patient-derived iPSC line could be useful in vitro tool for CMTX research as disease modeling and drug development.

Generation of induced pluripotent stem cell (iPSC) line from Charcot-Marie-Tooth disease patient with MPZ mutation (CMT1B).

Charcot-Marie-Tooth disease (CMT1B) is an inherited neurological disorder caused by mutation of the myelin protein zero (MPZ) gene. We generated an induced pluripotent stem cell (iPSC) line from an 81-year-old patient with CMT1B by electroporating of lymphoblastoid cell lines with episomal plasmids encoding OCT4, SOX2, KLF4, L-MYC, LIN28, and p53-targeting shRNA. The established iPSCs expressed various pluripotency markers, demonstrated the potential to differentiate into cells of the three germ layers in vitro, had a normal karyotype and retained the MPZ mutation.

Generation of two induced pluripotent stem cell (iPSC) lines from X-linked adrenoleukodystrophy (X-ALD) patients with adrenomyeloneuropathy (AMN).

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by a mutation in the ATP-binding cassette transporter subfamily D member 1 (ABCD1) gene. We generated two induced pluripotent stem cell (iPSC) lines from X-ALD patients with adrenomyeloneuropathy (AMN) by Sendai virus containing OCT4, SOX2, KLF4 and c-MYC. Established iPSC lines expressed various pluripotency markers, had differentiation potential of three germ layers in vitro, had normal karyotype and retained ABCD1 mutation.

Generation of induced pluripotent stem cell (iPSC) line from a 21-year-old X-linked adrenoleukodystrophy (X-ALD) patient.

X-linked Adrenoleukodystrophy (X-ALD) is a genetic disease that caused by mutations in adenosine triphosphate [ATP]-binding-cassette transporter superfamily D member 1 (ABCD1) gene. We generated an induced pluripotent stem cell (iPSC) line from a 21-year-old male X-ALD patient-derived fibroblasts by Sendai virus mediated reprogramming. Established iPSCs stably expanded while maintaining immunoreactivity for various pluripotency markers and alkaline phosphatase, as well as normal 44+XY karyotype. Under the differentiation condition, the cells gave rise to cells of three germ layers.