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.

Generation of mutation-corrected induced pluripotent stem cell lines derived from adrenoleukodystrophy patient by using homology directed repair.

X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. Previously, we generated an ALD patient-derived SCHi001-A iPSC model. In this study, we have performed the first genome editing of ALD patient-derived SCHi001-A iPSCs using homology-directed repair (HDR). The mutation site, c.1534G > A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal iPSC plulipotency marker expression following genome editing. Mutation-corrected iPSCs from SCHi001-A iPSC line can be used in research into the pathophysiology of and therapeutics for ALD.

OCT4-induced oligodendrocyte progenitor cells promote remyelination and ameliorate disease.

The generation of human oligodendrocyte progenitor cells (OPCs) may be therapeutically valuable for human demyelinating diseases such as multiple sclerosis. Here, we report the direct reprogramming of human somatic cells into expandable induced OPCs (iOPCs) using a combination of OCT4 and a small molecule cocktail. This method enables generation of A2B5+ (an early marker for OPCs) iOPCs within 2 weeks retaining the ability to differentiate into MBP-positive mature oligodendrocytes. RNA-seq analysis revealed that the transcriptome of O4+ iOPCs was similar to that of O4+ OPCs and ChIP-seq analysis revealed that putative OCT4-binding regions were detected in the regulatory elements of CNS development-related genes. Notably, engrafted iOPCs remyelinated the brains of adult shiverer mice and experimental autoimmune encephalomyelitis mice with MOG-induced 14 weeks after transplantation. In conclusion, our study may contribute to the development of therapeutic approaches for neurological disorders, as well as facilitate the understanding of the molecular mechanisms underlying glial development.

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.

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.

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.

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.

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.

A study on decoding models for the reconstruction of hand trajectories from the human magnetoencephalography.

Decoding neural signals into control outputs has been a key to the development of brain-computer interfaces (BCIs). While many studies have identified neural correlates of kinematics or applied advanced machine learning algorithms to improve decoding performance, relatively less attention has been paid to optimal design of decoding models. For generating continuous movements from neural activity, design of decoding models should address how to incorporate movement dynamics into models and how to select a model given specific BCI objectives. Considering nonlinear and independent speed characteristics, we propose a hybrid Kalman filter to decode the hand direction and speed independently. We also investigate changes in performance of different decoding models (the linear and Kalman filters) when they predict reaching movements only or predict both reach and rest. Our offline study on human magnetoencephalography (MEG) during point-to-point arm movements shows that the performance of the linear filter or the Kalman filter is affected by including resting states for training and predicting movements. However, the hybrid Kalman filter consistently outperforms others regardless of movement states. The results demonstrate that better design of decoding models is achieved by incorporating movement dynamics into modeling or selecting a model according to decoding objectives.