Differentiation Maturation Research Articles

Construction of a rodent neural network-skeletal muscle assembloid that simulate the postnatal development of spinal cord motor neuronal network

Neuromuscular diseases usually manifest as abnormalities involving motor neurons, neuromuscular junctions, and skeletal muscle (SkM) in postnatal stage. Present in vitro models of neuromuscular interactions require a long time and lack neuroglia involvement. Our study aimed to construct rodent bioengineered spinal cord neural network-skeletal muscle (NN-SkM) assembloids to elucidate the interactions between spinal cord neural stem cells (SC-NSCs) and SkM cells and their biological effects on the development and maturation of postnatal spinal cord motor neural circuits. After coculture with SkM cells, SC-NSCs developed into neural networks (NNs) and exhibited a high proportion of glutamatergic and cholinergic neurons, low proportion of neuroglia and gamma-aminobutyric acidergic neurons, and increased expression of synaptic markers. In NN-SkM assembloids, the acetylcholine receptors of SkM cells were upregulated, generating neuromuscular junction-like structures with NNs. The amplitude and frequency of SkM cell contraction in NN-SkM assembloids were increased by optogenetic and glutamate stimulation and blocked by tetrodotoxin and dizocilpine, respectively, confirming the existence of multisynaptic motor NNs. The coculture process involves the secretion of neurotrophin-3 and insulin growth factor-1 by SkM cells, which activate the related ERK-MAPK and PI3K-AKT signaling pathways in NNs. Inhibition of the ERK-MAPK and PI3K-AKT pathways significantly reduces neuronal differentiation and synaptic maturation of neural cells in NN-SkM assembloids, while also decreasing acetylcholine receptor formation on SkM cells. In brief, NN-SkM assembloids simulate the composition of spinal cord motor NNs and respond to motor regulatory signals, providing an in vitro model for studying postnatal development and maturation of spinal cord motor NNs.

Adult Human Heart ECM Improves Human iPSC-CM Function via Mitochondrial and Metabolic Maturation.

Myocardial infarction can lead to the loss of billions of cardiomyocytes, and while cell-based therapies are an option, immature nature of in vitro-generated human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs) is a roadblock to their development. Existing iPSC differentiation protocols don't go beyond producing fetal iCMs. Recently, adult extracellular matrix (ECM) was shown to retain tissue memory and have some success driving tissue-specific differentiation in unspecified cells in various organ systems. Therefore, we focused on investigating the effect of adult human heart-derived ECM on iPSC cardiac differentiation and subsequent maturation. By preconditioning iPSCs with ECM, we tested whether creating cardiac environments around iPSCs would drive iPSCs toward cardiac fate and which ECM components might be involved. We report novel high- and low- abundance proteomes of young, adult, and aged human hearts, with relative abundances to total proteins and each other. We found that adult ECM had extracellular galactin-1, fibronectin, fibrillins, and perlecan (HSPG2) which are implicated in normal heart development. We also showed preconditioning iPSCs with adult cardiac ECM resulted in enhanced cardiac differentiation, yielding iCMs with higher functional maturity, more developed mitochondrial network and coverage, enhanced metabolic maturity, and shift towards more energetic profile. These findings demonstrate the potential use of cardiac ECM in iCM maturation and as a promising strategy for developing iCM-based therapies, disease modeling, and drug screening studies. Upon manipulating ECM, we concluded that the beneficial effects observed were not solely due to the ECM proteins, which might be related to the decorative units attached.

Effect of introducing somatic mitochondria into an early embryo on zygotic gene activation†.

Unlike differentiated somatic cells, which possess elongated mitochondria, undifferentiated cells, such as those of preimplantation embryos, possess round, immature mitochondria. Mitochondrial morphology changes dynamically during cell differentiation in a process called mitochondrial maturation. The significance of the alignment between cell differentiation and mitochondrial maturity in preimplantation development remains unclear. In this study, we analyzed mouse embryos into which liver-derived somatic mitochondria were introduced (SM-embryos). Most SM-embryos were arrested at the two-cell stage. Some of the introduced somatic mitochondria became round, while others remained elongated and large. RNA-sequencing revealed a disruption of both minor and major zygotic gene activation (ZGA) in SM-embryos. Minor ZGA did not terminate before major ZGA, and the onset of major ZGA was inhibited, as shown by histone modification analyses of histone H3 lysine 4 trimethylation and histone H3 lysine 27 acetylation. Further analysis of metabolites involved in histone modification regulation in SM-embryos showed a significantly lower NAD+/NADH ratio in SM-embryos than in control embryos. Additionally, the mitochondrial membrane potential, an indicator of mitochondrial function, was lower in SM-embryos than in control embryos. Our results demonstrated that introducing somatic mitochondria into an embryo induces mitochondrial dysfunction, thereby disrupting metabolite production, leading to a disruption in ZGA and inducing developmental arrest. Our findings reveal that the alignment between cell differentiation and mitochondrial maturity is essential for early embryonic development.

Audiovisual gamma stimulation enhances hippocampal neurogenesis and neural circuit plasticity in aging mice.

Gamma oscillations are disrupted in various neurological disorders, including Alzheimer's disease (AD). In AD mouse models, non-invasive audiovisual stimulation (AuViS) at 40 Hz enhances gamma oscillations, clears amyloid-beta, and improves cognition. We investigated mechanisms of circuit remodeling underlying these restorative effects by leveraging the sensitivity of hippocampal neurogenesis to activity in middle-aged wild-type mice. AuViS increased progenitor cell proliferation, neuronal differentiation and morphological maturation of newborn granule cells, promoting their synaptic integration. While visual or auditory stimuli alone induced dendritic growth, axonal changes required combined audiovisual stimulation. The actions of AuViS involved neurotrophin pathways, as shown by the lack of effect upon TrkB signaling blockade. These results reveal widespread plasticity mechanisms triggered by AuViS, a therapeutic approach currently proposed for treating neurological disorders in humans.

Histochemical indications for a chemically complex signal produced by the cervical gill slit gland of the pygmy sperm whale (Kogia breviceps).

The pygmy sperm whale (Kogia breviceps) possesses an exocrine gland associated with its false gill slit pigmentation pattern. The cervical gill slit gland is a compound tubuloalveolar gland that produces a holocrine secretion and displays maturational changes in size and secretory histology. While the morphology of the cervical gill slit gland has been described in detail, to date, the chemical composition of its secretion remains uncharacterized. This study used histochemical staining techniques and quantitative lipid analysis to identify and characterize the constituents expressed in the secretory cells and secretion of the cervical gill slit gland. Results demonstrate that the secretion, like those of terrestrial artiodactyls that function in chemical communication, includes a complex mixture of carbohydrates, proteins, and lipids. Differences in staining intensity across germinal and secretory epithelial layers demonstrate differential expression, or maturation, of mucins and proteins. Additionally, a highly unusual and primary constituent of the secretion is uric acid. Uric acid was identified within the secretion using histochemical stains and polarized light imaging, and chemically verified using scanning electron microscopy with energy dispersive spectrometry. While uric acid is not a common constituent of mammalian exocrine glands, urate-based compounds are abundant in the secretions of marine organisms used in chemical communication. Thus, uric acid may contribute to the chemical message produced by K. breviceps in its marine environment. We hypothesize that the chemical signals produced by the gill slit gland may be shared at close-range by conspecifics, and that the mode of sensory reception is likely gustation.

Alterations in cellular metabolic pathway and epithelial cell maturation induced by MYO5B defects are partially reversible by LPAR5 activation.

Functional loss of the motor protein myosin Vb (MYO5B) induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, namely, microvillus inclusion disease (MVID). Utilizing the MVID model mice Vil1-CreERT2;Myo5bflox/flox (MYO5BΔIEC) and Vil1-CreERT2;Myo5bflox/G519R [MYO5B(G519R)], we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in the MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5BΔIEC small intestine. Consistent with these observations in vivo, RNA sequencing of enteroids generated from the two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, we reported that lysophosphatidic acid (LPA) signaling ameliorated epithelial cell defects in MYO5BΔIEC tissues and enteroids. The present study demonstrated that the highly soluble LPA receptor (LPAR)5-preferred agonist Compound-1 improved sodium transporter localization and absorptive function and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on the intestine with MYO5B functional loss through epithelial cell-autonomous pathways that can accelerate the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for the treatment of MVID symptoms induced by different point mutations in MYO5B.NEW & NOTEWORTHY This study demonstrates the importance of MYO5B for cellular lipid metabolism and mitochondria in intestinal epithelial cells, previously unexplored functions of MYO5B. The alterations may underlie the progenitor cell malfunction observed in microvillus inclusion disease (MVID) intestines. To examine the therapeutic potential of progenitor-targeted treatments, the effects of the LPAR5-preferred agonist Compound-1 were investigated utilizing several MVID model mice and enteroids. Our observations suggest that Compound-1 may provide a therapeutic approach for treating MVID.

Palovarotene (Sohonos), a synthetic retinoid for reducing new heterotopic ossification in fibrodysplasia ossificans progressiva: history, present, and future.

Retinoids are metabolic derivatives of vitamin A and play crucial roles in the regulation of various tissues and organs during prenatal and postnatal development. Active retinoids, like all-trans-retinoic acid, are synthesized in the cytoplasm and subsequently interact with nuclear retinoic acid receptors (RARα, RARβ, and RARγ) to enhance transcription of specific genes. In the absence of retinoids, RARs can still bind to response elements of target genes but repress their transcription. Chondrogenic cell differentiation and cartilage maturation in the growth plate require the absence of retinoid signaling and transcriptional repression by unliganded RARs. This led to the hypothesis that synthetic retinoid agonists may be pharmacological agents to inhibit those cellular processes and counter the excessive formation of cartilage and bone in conditions like heterotopic ossification (HO). HO can be instigated by diverse culprits including trauma, invasive surgeries, inflammatory disorders, or genetic conditions. One such genetic disease is fibrodysplasia ossificans progressiva (FOP), a rare disorder driven by activating mutations in the ACVR1 gene. Patients with FOP have severe and progressive HO formation in soft tissues, leading to extensive permanent loss of mobility and increased mortality. Synthetic retinoid agonists selective for RARα or RARγ showed efficacy against injury-induced and genetic HO in mouse models. The RARγ agonists showed the highest effectiveness, with palovarotene being selected for clinical trials in patients with FOP. Post hoc analyses of phase II and phase III clinical trials showed that palovarotene has significant disease-modifying effects for FOP, but with significant risks such as premature growth plate closure in some younger subjects. This review provides an overview of retinoid and RAR roles in skeletal development and discusses the identification of palovarotene as a potential FOP therapy, the clinical data supporting its regulatory approval in some countries, and the potential applications of this drug for other relevant disorders besides FOP.

Controlling spheroid attachment improves pancreatic beta cell differentiation from human iPS cells.

Regenerative medicine using human induced pluripotent stem cells (hiPSCs) is available for treating type 1 diabetes; however, the efficiency and maturation of hiPSC differentiation into pancreatic beta cells requires improvement. Various protocols, including three-dimensional (3D) culture, have been developed to improve differentiation efficiency and maturation. Several methods for 3D culture have been reported; however, they require costly and complicated equipment, special materials, and complicated operations. To solve these problems, we developed a simple 3D culture method under static conditions using a cyclo-olefin polymer (COP) characterized by high moisture barrier properties, low surface energy, and hydrophobicity. Using this 3D method and our simple and low-cost protocol, we found that differentiation into the definitive endoderm (DE) was better when the spheroids were attached. Therefore, upon the addition of Y-27632, attached spheroids with unique shapes and cavities were formed, and the differentiation efficiency into DE increased. During DE differentiation, the attachment of spheroids to the substrate and their subsequent floating improved differentiation efficiency. We found that the amount of C-peptide in spheroids differentiated using COP dishes was greater than that in rotary culture. Furthermore, INSULIN was highly expressed in areas with low cell density, suggesting that the unique shape of the spheroids made from COP dishes improved differentiation efficiency. Our study suggests that a device-free, simple 3D culture method that controls spheroid attachment improves the efficiency of hiPSC differentiation into pancreatic beta cells.

Maternal early overfeeding negatively impacts cardiac progenitor cells differentiation and cardiomyocyte maturation in the neonatal offspring.

Maternal obesity has been positively correlated with an increased cardiometabolic risk in the offspring throughout life, implying intergenerational transmission. However, little is known about the early life cardiac cell modifications that imply the onset of heart diseases later in life. This study analyzed cardiac progenitor cells and cardiomyocyte differentiation on day of birth in the offspring born to obese dams. The litter size reduction model was used to induce obesity in female Swiss mice. Both maternal groups, the Small Litter Dams (SLD-F1), which were overfed during lactation, and the Normal Litter Dams (NLD-F1), control group, were mated to healthy male mice. Their first generation offspring (SLD-F2 and NLD-F2, n=6 by group) were euthanized on birth. Mothers from SLD had increased body mass, Lee Index, fat deposits, hyperglycemia, and glucose intolerance, confirming the obese phenotype. The offspring born from SLD-F1 had also increased body mass, Lee Index, and fasting hyperglycemia. The heart of SLD-F2 showed decreased cardiac mass/body mass ratio, increased cardiac collagen deposits, and a greater number of undifferentiated cardiac c-kit+ and Sca-1+ progenitor cells, and increased NKX2.5+ cardiomyoblasts compared to control. In addition, SLD-F2 demonstrated immature cardiomyocytes. Obese dams negatively impact its offspring, leading to altered biometric and metabolic parameters, along with an immature heart already at birth, with extracellular matrix adverse remodeling, delayed cardiac progenitor cells differentiation and restrained cardiomyocyte maturation, which can be related with the development of cardiometabolic disease in the adulthood.

Placenta‐Derived Mesenchymal Stromal‐Like Cells Promote 3D‐Engineered Muscle Tissue Differentiation and Vessel Network Maturation

Placenta‐Derived Mesenchymal Stromal‐Like Cells Promote 3D‐Engineered Muscle Tissue Differentiation and Vessel Network Maturation

Synergistic effects of human umbilical cord mesenchymal stem cells/neural stem cells and epidural electrical stimulation on spinal cord injury rehabilitation

Spinal cord injury (SCI) is a severe neurological condition marked by a complex pathology leading to irreversible functional loss, which current treatments fail to improve. Epidural electrical stimulation (EES) shows promise in alleviating pathological pain, regulating hemodynamic disturbances, and enhancing motor function by modulating residual interneurons in the lower spinal cord. Cell transplantation (CT), especially using human umbilical cord mesenchymal stem cells (hUCMSCs) and neural stem cells (NSCs), has significantly improved sensory and motor recovery in SCI. However, the limitations of single treatments have driven the exploration of a multifaceted strategy, combining various modalities to optimize recovery at different stages. To comprehensively investigate the effectiveness of in situ transplantation of hUCMSCs/NSCs combined with subacute epidural electrical stimulation in a murine spinal cord crush injury model, providing valuable references for future animal studies and clinical research. In this study, we first examined neural stem cell changes via mRNA sequencing in an in vitro Transwell co-culture model. We then explored cell interaction mechanisms using proliferation assays, differentiation assays, and neuron complexity analysis. For animal experiments, 40 C57BL/6 mice were assigned to four groups (Injury/EES/CT/Combination). Histological evaluations employed HE and immunofluorescence staining, while electrophysiological and behavioral tests assessed motor recovery. Quantitative data were reported as mean ± standard error, with statistical analyses performed using GraphPad Prism and SPSS. Initially, we found that NSCs in the in vitro co-culture model showed a unique expression profile of differentially expressed genes (DEGs) compared to controls. GO/KEGG analysis indicated these DEGs were mainly linked to cell differentiation and growth factor secretion pathways. Neuronal and astrocytic markers further confirmed enhanced NSC differentiation and neuronal maturation in the co-culture model. In vivo, live imaging and human nuclei immunofluorescence staining revealed that transplanted cells persisted for some time post-transplantation. Histological analysis showed that during acute inflammation, both the stem cell and combined therapy groups significantly inhibited microglial polarization. In the chronic phase, these groups reduced fibrotic scar formation and encouraged astrocytic bridging. Behavioral tests, including swimming and gait analysis, demonstrated that combined CT and EES therapy was more effective than either treatment alone. In summary, the combined therapy offers a promising approach for spinal cord injury treatment, providing superior outcomes over individual treatments. Our findings underscore the potential of a combined treatment approach utilizing stem cells transplantation and EES as an effective strategy for the comprehensive management of spinal cord crush injury in mice. This integrated approach holds promise for enhancing functional recovery and improving the quality of life for individuals with spinal cord injury (SCI).

Neuritin alleviates Diabetic Retinopathy by Regulating Endoplasmic Reticulum Stress in Rats.

Neuritin, a small-molecule neurotrophic factor, maintains neuronal cell activity, inhibits apoptosis, promotes process growth, and regulates neural progenitor cell differentiation, migration, and synaptic maturation. Neuritin helps retinal ganglion cells (RGCs) survive optic nerve injury in rats and regenerate axons. However, the role of Neuritin in Diabetic retinopathy (DR) is unclear. This study is intended to investigate the effect and mechanism of Neuritin in DR. For this purpose, we established DR rat models and injected Neuritin into them. This study provides a potential treatment for diabetic retinopathy. The rat model of DR was established by streptozotocin (STZ) injection, and the effect of Neuritin on DR was detected by intravitreal injection. Histological analysis was performed by H&E and TUNEL methods. The mRNA and protein expressions of endoplasmic reticulum stress (ERS) pathway-related transcription factors were detected by qRT-PCR and western blot. The blood-retinal barrier (BRB) function was assessed using the patch-clamp technique and Evans blue leakage assay. Neuritin significantly improved the retinal structure, restrained the apoptosis of retinal cells, and protected the normal function of BRB in DR model rats. Mechanistically, Neuritin may function by inhibiting the expression of GRP78, ASK1, Caspase-12, VEGF, and so on. Our results indicate that Neuritin alleviates retinal damage in DR rats via the inactive endoplasmic reticulum pathway. Our study provides a potential treatment for DR.

Generation of ex vivo autologous hematopoietic stem cell-derived T lymphocytes for cancer immunotherapy

Generation of ex vivo autologous hematopoietic stem cell-derived T lymphocytes for cancer immunotherapy

Genome-wide association study of growth and reproductive traits based on low-coverage whole-genome sequencing in a Chubao black-head goat population

The Chubao black-head goat is a novel hybrid breed that combines the advantages of Macheng black goats, such as good reproductive performance, strong adaptability, and resistance to rough feeding, with the superior growth and meat characteristics of Boer goats. Given the substantial economic importance of growth (such as birth weight, body height, body length, and chest circumference across different growth stages) and reproductive traits (particularly average litter size after first parity), the aim of this study was to identify significant SNPs and candidate genes associated with these traits in Chubao black-head goats. Through whole-genome sequencing (with 34 goats at approximately 15× coverage and 466 goats at approximately 1× coverage), genotype imputation, and quality control, 22,665,331 SNPs were identified and subsequently used for genetic analyses. Heritability estimates indicated that growth traits exhibit moderate to high heritability (ranging from 0.297±0.071 to 0.535±0.118), while reproductive traits demonstrated low to moderate heritability (with a value of 0.220±0.108). By performing FarmCPU-based genome-wide association studies, we identified 48 potentially significant SNPs associated with growth traits and 7 with reproductive traits. Additionally, 85 candidate genes (such as COL14A1, ZNF148, and TTC39C) linked to growth traits were identified and enriched in pathways associated with fundamental molecular biological activities such as protein deubiquitination, regulation of mRNA stability, and the MAPK signaling pathway. Furthermore, 10 candidate genes (such as SOHLH2, CCNA2, and SOX7) associated with reproductive traits were identified and enriched in pathways related to specific reproductive processes such as oocyte differentiation, endoderm formation, and progesterone-mediated oocyte maturation. Overall, these findings provide valuable preliminary insights into the molecular mechanisms underlying growth and reproductive traits in Chubao black-head goats. However, further functional validation is needed to effectively use these potential SNPs and candidate genes in improving the breeding of these traits in this breed.

Myotube formation on micropatterns guiding by centripetal cellular motility and crowding

The physical microenvironment, including substrate rigidity and topology, impacts myoblast differentiation and myotube maturation. However, the interplay effect and physical mechanism of mechanical stimuli on myotube formation is poorly understood. In this study, we utilized elastic substrates, microcontact patterning technique, and particle image velocimetry to investigate the effect of substrate rigidity and topological constraints on myoblast behaviors. Our findings suggested the interplay of substrate stiffness and cellular confinement improved the myotube formation by inducing centripetal cellular motility. These results shed light on the impact of the topological substrate on myoblast differentiation and emphasize the critical role of asymmetrical cell motility during this process, which is highly correlated with cell movement and crowding. Our research provides insights into the intricate interplay between substrate properties, cell motility, and myotube formation during myogenesis. Understanding these mechanisms could trigger tissue engineering strategies and therapies to enhance muscle regeneration and function.

Placenta‐Derived Mesenchymal Stromal‐Like Cells Promote 3D‐Engineered Muscle Tissue Differentiation and Vessel Network Maturation

Placental‐derived stromal‐like cells (PLX‐PAD) have been shown to facilitate muscle tissue recovery after injury and stimulate angiogenesis. This work assesses the impact of PLX‐PAD cells on the vascularization and maturation of engineered skeletal muscle tissue. Specifically, their effects in direct co‐culture with endothelial cells, pericytes, and myoblasts seeded within microporous 3D scaffolds are characterized. Additionally, the impact of hypoxic PLX‐PAD cell‐conditioned medium (CM) on vascularization and muscle differentiation of engineered tissue is monitored. Co‐culture of PLX‐PAD with myocytes stimulated myocyte differentiation while PLX‐PAD CM promoted the formation of vascular networks. Implantation of a multi‐culture system of vascularized human skeletal muscle tissue and PLX‐PAD into a rectus abdominal defect in nude mice promoted myocyte differentiation, host vessel penetration, and tissue integration. These findings indicate the ability of placenta‐derived cells to induce the formation of vascularized engineered muscle constructs with potential therapeutic applications.

Metabolic reprogramming and heterogeneity during the decidualization process of endometrial stromal cells

The human endometrial decidualization is a transformative event in the pregnant uterus that involves the differentiation of stromal cells into decidual cells. While crucial to the establishment of a successful pregnancy, the metabolic characteristics of decidual cells in vivo remain largely unexplored. Here, we integrated the single-cell RNA sequencing (scRNA-seq) datasets on the endometrium of the menstrual cycle and the maternal-fetal interface in the first trimester to comprehensively decrypt the metabolic characteristics of stromal fibroblast cells. Our results revealed that the differentiation of stromal cells into decidual cells is accompanied by increased amino acid and sphingolipid metabolism. Furthermore, metabolic heterogeneity exists in decidual cells with differentiation maturity disparities. Decidual cells with high metabolism exhibit higher cellular activity and show a strong propensity for signaling. In addition, significant metabolic reprogramming in amino acids and lipids also occurs during the transition from non-pregnancy to pregnancy in the uteri of pigs, cattle, and mice. Our analysis provides comprehensive insights into the dynamic landscape of stromal fibroblast cell metabolism, contributing to our understanding of the metabolism at the molecular dynamics underlying the decidualization process in the human endometrium.

Thickening tissue by thinning electrospun scaffolds for skeletal muscle tissue engineering

AbstractElectrospun scaffolds with aligned fiber orientation are widely used in tissue engineering, such as muscle, heart, nerve, tendon, and cartilage, due to their ability to guide cell morphology and induce cellular functions. However, the dense fibrous structure of the scaffolds poses a critical obstacle to engineering highly cellular and thick 3D tissues, as it prevents cell infiltration. While many techniques have been developed to increase the pore size of electrospun scaffolds and improve cell infiltration/migration, it often leads to a decrease in direct cell‐cell contact, compromising cell differentiation and tissue maturation. This study presents an alternative approach by reducing the thickness of scaffolds to the cellular scale and stacking or rolling the cell‐scaffold complex into 3D constructs. We devise a series of novel tools to fabricate, characterize, and manipulate ultra‐thin electrospun scaffolds, which demonstrate high reproducibility, resolution, and cellularity. Our study provides a solution to the cell infiltration issue in muscle tissue engineering and is highly versatile, and can be applied to various fields that require structures with high‐resolution gradients in a layered pattern or complex spatial distribution in a rolled pattern.

The hiPSC-derived corneal endothelial progenitor-like cell recovers the rabbit model of corneal endothelial dystrophy

Corneal endothelial dysfunction results in cornea opacity, damaging sightedness, and affecting quality of life. A corneal transplant is the current effective intervention. Due to the scarcity of donated cornea, such an unmet medical need requires a novel therapeutic modality. Customizing patients' corneal endothelial progenitor cells with proliferative activity and lineage restriction properties shall offer sufficient therapeutic cells for corneal endothelial dystrophy. The customized induced human corneal endothelial progenitor-like cell (iHCEPLC) was obtained through cell fate conversions starting from PBMC (peripheral blood mononuclear cell), hiPSC (human induced pluripotent stem cell), and hNCC (human neural crest cell), while it finally reached the iHCEPLC state via a series of induction. Several molecular diagnoses were applied to depict its progenitor state, including RNAseq, FlowCytometer, immunostainings, and rtPCR. Significantly, it can be induced to gain differentiation maturity through contact inhibition. In addition, a BAK-mediated rabbit model of corneal endothelial dystrophy was established in the present study to test the therapeutic effectiveness of the iHCEPLC. After inducing cell fate conversion, the specific HCEC markers were detected by rtPCR and immunostaining in iHCEPLC. Further, RNAseq was applied to distinguish its progenitor-like cell fate from primary human corneal endothelial cells (HECE). FlowCytometry profiled the heterogeneity subpopulation, consistently displaying a subtle difference from primary HCEC. A terminal differentiation can be induced in iHCEPLC, addressing its progenitor-like fate. iHCEPLC can restore the BAK-based rabbit model of corneal endothelial dystrophy. Immunohistochemistry verified that such acuity restoration of the BAK-treated cornea is due to the introduced iHCEPLC, and such therapeutic effectiveness is observed in the long term. Here, we demonstrated that customized iHCEPLC has long-term therapeutic efficacy. As a progenitor cell, our iHCEPLC has a restricted cell lineage nature and can proliferate in vitro, supporting sufficient therapeutic candidate cells. Due to the immune-privileged nature of the cornea, our iHCEPLC proves the principle of therapeutical feasibility in both autogenic and allogeneic modalities.

Characterization of TGFβ1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-β (TGFβ) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFβ signaling in tendon prompted us to utilize TGFβ1 to induce tendon-like structures in 3D tendon constructs. TGFβ1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFβ1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFβ1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.