Improving motor neuron-like cell differentiation of hEnSCs by the combination of epothilone B loaded PCL microspheres in optimized 3D collagen hydrogel.

Spinal cord regeneration is limited due to various obstacles and complex pathophysiological events after injury. Combination therapy is one approach that recently garnered attention for spinal cord injury (SCI) recovery. A composite of three-dimensional (3D) collagen hydrogel containing epothilone B (EpoB)-loaded polycaprolactone (PCL) microspheres (2.5 ng/mg, 10 ng/mg, and 40 ng/mg EpoB/PCL) were fabricated and optimized to improve motor neuron (MN) differentiation efficacy of human endometrial stem cells (hEnSCs). The microspheres were characterized using liquid chromatography-mass/mass spectrometry (LC-mas/mas) to assess the drug release and scanning electron microscope (SEM) for morphological assessment. hEnSCs were isolated, then characterized by flow cytometry, and seeded on the optimized 3D composite. Based on cell morphology and proliferation, cross-linked collagen hydrogels with and without 2.5 ng/mg EpoB loaded PCL microspheres were selected as the optimized formulations to compare the effect of EpoB release on MN differentiation. After differentiation, the expression of MN markers was estimated by real-time PCR and immunofluorescence (IF). The collagen hydrogel containing the EpoB group had the highest HB9 and ISL-1 expression and the longest neurite elongation. Providing a 3D permissive environment with EpoB, significantly improves MN-like cell differentiation and maturation of hEnSCs and is a promising approach to replace lost neurons after SCI.

Adult mesenchymal stem cell ageing interplays with depressed mitochondrial Ndufs6.

Mesenchymal stem cell (MSC)-based therapy has emerged as a novel strategy to treat many degenerative diseases. Accumulating evidence shows that the function of MSCs declines with age, thus limiting their regenerative capacity. Nonetheless, the underlying mechanisms that control MSC ageing are not well understood. We show that compared with bone marrow-MSCs (BM-MSCs) isolated from young and aged samples, NADH dehydrogenase (ubiquinone) iron-sulfur protein 6 (Ndufs6) is depressed in aged MSCs. Similar to that of Ndufs6 knockout (Ndufs6-/-) mice, MSCs exhibited a reduced self-renewal and differentiation capacity with a tendency to senescence in the presence of an increased p53/p21 level. Downregulation of Ndufs6 by siRNA also accelerated progression of wild-type BM-MSCs to an aged state. In contrast, replenishment of Ndufs6 in Ndufs6-/--BM-MSCs significantly rejuvenated senescent cells and restored their proliferative ability. Compared with BM-MSCs, Ndufs6-/--BM-MSCs displayed increased intracellular and mitochondrial reactive oxygen species (ROS), and decreased mitochondrial membrane potential. Treatment of Ndufs6-/--BM-MSCs with mitochondrial ROS inhibitor Mito-TEMPO notably reversed the cellular senescence and reduced the increased p53/p21 level. We provide direct evidence that impairment of mitochondrial Ndufs6 is a putative accelerator of adult stem cell ageing that is associated with excessive ROS accumulation and upregulation of p53/p21. It also indicates that manipulation of mitochondrial function is critical and can effectively protect adult stem cells against senescence.

Mitochondria-enriched protrusions are associated with brain and intestinal stem cells in Drosophila.

Brain stem cells stop dividing in late Drosophila embryos and begin dividing again in early larvae after feeding induces reactivation. Quiescent neural stem cells (qNSCs) display an unusual cytoplasmic protrusion that is no longer present in reactivated NSCs. The protrusions join the qNSCs to the neuropil, brain regions that are thought to maintain NSCs in an undifferentiated state, but the function of the protrusions is not known. Here we show that qNSC protrusions contain clustered mitochondria that are likely maintained in position by slow forward-and-backward microtubule growth. Larvae treated with a microtubule-stabilizing drug show bundled microtubules and enhanced mitochondrial clustering in NSCs, together with reduced qNSC reactivation. We further show that intestinal stem cells contain mitochondria-enriched protrusions. The qNSC and intestinal stem-cell protrusions differ from previously reported cytoplasmic extensions by forming stem-cell-to-niche mitochondrial bridges that could potentially both silence genes and sense signals from the stem cell niche.

Correlation between Nuclear Morphology and Adipogenic Differentiation: Application of a Combined Experimental and Computational Modeling Approach.

Stem cells undergo drastic morphological alterations during differentiation. While extensive studies have been performed to examine the cytoskeletal remodeling, there is a growing interest to determine the morphological, structural and functional changes of the nucleus. The current study is therefore aimed at quantifying the extent of remodeling of the nuclear morphology of human mesenchymal stem cells during biochemically-induced adipogenic differentiation. Results show the size of nuclei decreased exponentially over time as the lipid accumulation is up-regulated. Increases in the lipid accumulation appear to lag the nuclear reorganization, suggesting the nuclear deformation is a prerequisite to adipocyte maturation. Furthermore, the lamin A/C expression was increased and redistributed to the nuclear periphery along with a subsequent increase in the nuclear aspect ratio. To further assess the role of the nucleus, a nuclear morphology with a high aspect ratio was achieved using microcontact-printed substrate. The cells with an elongated nuclear shape did not efficiently undergo adipogenesis, suggesting the cellular and nuclear processes associated with stem cell differentiation at the early stage of adipogenesis cause a change in the nuclear morphology and cannot be abrogated by the morphological cues. In addition, a novel computational biomechanical model was generated to simulate the nuclear shape change during differentiation and predict the forces acting upon the nucleus. This effort led to the development of computational scaling approach to simulate the experimentally observed adipogenic differentiation processes over 15 days in less than 1.5 hours.

Adult stem cell deficits drive Slc29a3 disorders in mice.

Mutations exclusively in equilibrative nucleoside transporter 3 (ENT3), the only intracellular nucleoside transporter within the solute carrier 29 (SLC29) gene family, cause an expanding spectrum of human genetic disorders (e.g., H syndrome, PHID syndrome, and SHML/RDD syndrome). Here, we identify adult stem cell deficits that drive ENT3-related abnormalities in mice. ENT3 deficiency alters hematopoietic and mesenchymal stem cell fates; the former leads to stem cell exhaustion, and the latter leads to breaches of mesodermal tissue integrity. The molecular pathogenesis stems from the loss of lysosomal adenosine transport, which impedes autophagy-regulated stem cell differentiation programs via misregulation of the AMPK-mTOR-ULK axis. Furthermore, mass spectrometry-based metabolomics and bioenergetics studies identify defects in fatty acid utilization, and alterations in mitochondrial bioenergetics can additionally propel stem cell deficits. Genetic, pharmacologic and stem cell interventions ameliorate ENT3-disease pathologies and extend the lifespan of ENT3-deficient mice. These findings delineate a primary pathogenic basis for the development of ENT3 spectrum disorders and offer critical mechanistic insights into treating human ENT3-related disorders.

Multipotent Adult Progenitor Cells Suppress T Cell Activation in In Vivo Models of Homeostatic Proliferation in a Prostaglandin E2-Dependent Manner.

Lymphodepletion strategies are used in the setting of transplantation (including bone marrow, hematopoietic cell, and solid organ) to create space or to prevent allograft rejection and graft versus host disease. Following lymphodepletion, there is an excess of IL-7 available, and T cells that escape depletion respond to this cytokine undergoing accelerated proliferation. Moreover, this environment promotes the skew of T cells to a Th1 pro-inflammatory phenotype. Existing immunosuppressive regimens fail to control this homeostatic proliferative (HP) response, and thus the development of strategies to successfully control HP while sparing T cell reconstitution (providing a functioning immune system) represents a significant unmet need in patients requiring lymphodepletion. Multipotent adult progenitor cells (MAPC®) have the capacity to control T cell proliferation and Th1 cytokine production. Herein, this study shows that MAPC cells suppressed anti-thymocyte globulin-induced cytokine production but spared T cell reconstitution in a pre-clinical model of lymphodepletion. Importantly, MAPC cells administered intraperitoneally were efficacious in suppressing interferon-γ production and in promoting the expansion of regulatory T cells in the lymph nodes. MAPC cells administered intraperitoneally accumulated in the omentum but were not present in the spleen suggesting a role for soluble factors. MAPC cells suppressed lymphopenia-induced cytokine production in a prostaglandin E2-dependent manner. This study suggests that MAPC cell therapy may be useful as a novel strategy to target lymphopenia-induced pathogenic T cell responses in lymphodepleted patients.

Ghrelin protects adult rat hippocampal neural stem cells from excessive autophagy during oxygen-glucose deprivation.

Ghrelin functions as a neuroprotective agent and saves neurons from various insults include ischemic injury. However, it remains to be elucidated whether ghrelin protects neuronal cells against ischemic injury-induced excessive autophagy. Autophagy is required for the maintenance of neural stem cell homeostasis. However, regarding autophagic cell death, it is commonly assumed that excessive autophagy leads to self-elimination of mammalian cells. The purpose of this study was to investigate the potential neuroprotection effects of ghrelin from excessive autophagy in adult rat hippocampal neural stem cells (NSCs). Oxygen-Glucose Deprivation (OGD) strongly induces autophagy in adult rat hippocampal NSCs. Ghrelin treatment inhibited OGD-induced cell death of adult rat hippocampal NSCs assessed by cell-counting-kit-8 assay. Ghrelin also suppressed OGD-induced excessive autophagy activity. The protective effect of ghrelin was accompanied by an increased expression levels of Bcl-2, p-62 and decreased expression level of LC3-II, Beclin-1 by Western blot. Furthermore, ghrelin reduced autophagosome formation and number of GFP-LC3 transfected puncta. In conclusion, our data suggest that ghrelin protects adult rat hippocampal NSCs from excessive autophagy in experimental stroke (oxygen-glucose deprivation) model. Regulating autophagic activity may be a potential optimizing target for promoting adult rat hippocampal NSCs based therapy for stroke.

Collagen and Hydroxyapatite Scaffolds Activate Distinct Osteogenesis Signaling Pathways in Adult Adipose-Derived Multipotent Stromal Cells.

Osteogenic cell signaling pathway disruption varies among bone diseases. This investigation was designed to identify adipose-derived multipotent stromal cell (ASC) and bone graft scaffold combinations for local, targeted restoration of gene expression and extracellular matrix (ECM) deposition. Human ASC osteogenesis on bone graft materials was quantified following culture in stromal (S), osteogenic (O), or osteogenic for 48 h followed by stromal medium (OS) to test the two-part hypothesis: (1) identical ASC isolates on distinct bone graft scaffolds demonstrate unique viability, differentiation, ECM production, and gene expression in the same culture conditions; (2) identical ASC-bone graft scaffold combinations have different cell viability, differentiation, ECM production, and gene expression when cultured in S, O, or OS medium. Three commercially available bone graft scaffold materials, type I bovine collagen (C), hydroxyapatite + ß-tricalcium phosphate + type I bovine collagen (HT), and ß-tricalcium phosphate + type I bovine collagen (CT) were evaluated. Passage 3 ASCs were loaded onto scaffold blocks with a spinner flask bioreactor, and constructs were cultured up to 28 days. Cell viability, gene expression (alkaline phosphatase [ALPL], osteoprotegerin [TNFRSF11B], osteocalcin [BGLAP], cannabinoid receptors type I [CNR1] and II [CNR2], receptor activator of nuclear factor kappa ß ligand [TNFSF11]), as well as ECM DNA, collagen, sulfated glycosaminoglycan, and protein content were quantified. Matrix organization was evaluated with scanning electron microscopy. Effects of scaffold, medium, or culture duration on cell viability were minimal. Significantly higher initial ALPL expression decreased with time, while BGLAP expression increased in HT constructs in O medium, and the constructs had the most abundant ECM components and ultrastructural organization. There was a similar, although delayed, pattern of gene expression and greater ECM collagen with less organization in C constructs in O medium. Higher CNR1 expression in C versus higher TNFRSF11B/TNFSF11 expression in HT constructs throughout the study support stimulation of unique osteogenic signaling pathways by identical cell isolates. These results suggest that bone scaffold composition may be used to selectively target specific osteogenic cell signaling pathways in ASC constructs to stimulate ECM deposition based on therapeutic needs.

Reovirus safety study for proliferation and differentiation of human adipose-derived mesenchymal stem cells.

Naturally occurring reoviruses are live replication-proficient viruses specifically infecting human cancer cells while sparing the normal counterparts. Stem cells can be highly susceptible to viral infection due to their innate high proliferation potential and other active signaling pathways of cells that might be involved in viral tropism. In the previous study, we showed that reoviruses could adversely affect murine embryonic stem cells' integrity in vitro and in vivo. Oncolytic viruses, delivered systemically face many hurdles that also impede their localization and infection of, metastatic tumors, due to a variety of immune and physical barriers. To overcome such hurdles to systemic delivery, several studies supported the idea that certain types of cells, including mesenchymal stem cells, might play a role as cell carriers for oncolytic viruses. Thus, it would be interesting to examine whether human adult stem cells such as human adipose-derived mesenchymal stem cells could be saved by the reoviral challenge. In this study, we report that biological activities such as proliferation and multipotency of human adipose-derived stem cells are not affected by wild-type reovirus challenge as evidenced by survival, osteogenic and adipogenic differentiation potential assays following treatment with reoviruses. Therefore, unlike murine embryonic stem cells, our study strongly suggests that human adipose-derived adult stem cells could be spared in vivo during wild-type reoviral anti-cancer therapeutics in a clinical setting. Furthermore, the results support the possible clinical use of human adipose-derived stem cells as an effective cell carrier of oncolytic reovirus to maximize their tumor tropism and anti-tumor activity.

The expansion of adult stem/progenitor cells and their marker expression fluctuations are linked with pituitary plastic adaptation during gestation and lactancy.

Extensive evidence has revealed variations in the number of hormone-producing cells in the pituitary gland, which occur under physiological conditions such as gestation and lactancy. It has been proposed that new hormone-producing cells differentiate from stem cells. However, exactly how and when this takes place is not clear. In this work, we used immunoelectron microscopy to identify adult pituitary stem/progenitor cells (SC/P) localized in the marginal zone (MZ), and additionally, we detected GFRa2-, Sox2-, and Sox9-positive cells in the adenoparenchyma (AP) by fluorescence microscopy. Then, we evaluated fluctuations of SC/P mRNA and protein level markers in MZ and AP during gestation and lactancy. An upregulation in stemness markers was shown at term of gestation (AT) in MZ, whereas there were more progenitor cell markers in the middle of gestation and active lactancy. Concerning committed cell markers, we detected a rise in AP at beginning of lactancy (d1L). We performed a BrdU uptake analysis in MZ and AP cells. The highest level of BrdU uptake was observed in MZ AT cells, whereas in AP this was detected in d1L, followed by a decrease in both the MZ and AP. Finally, we detected double immunostaining for BrdU-GFRa2 in MZ AT cells and BrdU-Sox9 in the AP d1L cells. Taken together, we hypothesize that the expansion of the SC/P niche took place mainly in MZ from pituitary rats in AT and d1L. These results suggest that the SC niche actively participates in pituitary plasticity during these reproductive states, contributing to the origin of hormone cell populations.

Oriented nanofibrous silk as a natural scaffold for ocular epithelial regeneration.

The aim of this study was to develop nanofibrous silk substrates for limbal stem cell expansion that can serve as a potential alternative substrate to replace human amniotic membrane. The human limbal stem cell was used to evaluate the biocompatibility of substrates (random and oriented nanofibrous mats, and human amniotic membrane) based on their phenotypic profile, viability, proliferation, and attachment ability. Biocompatibility results indicated that all substrates were highly biocompatible, as limbal stem cells could favorably attach and proliferate on the nanofibrous surfaces. Microscopic figures showed that the human limbal stem cells were firmly anchored to the substrates and were able to retain a normal corneal stem cell phenotype. Microscopic analyses illustrated that cells infiltrated the nanofibers and successfully formed a three-dimensional corneal epithelium, which was viable for 15 days. Immunocytochemistry and real-time PCR results revealed no change in the expression profile of limbal stem cells grown on nanofibrous substrates when compared to those grown on human amniotic membrane. In addition, electrospun nanofibrous silk substrates especially oriented mat provides not only a milieu supporting limbal stem cells expansion, but also serve as a useful alternative carrier for ocular surface tissue engineering and could be used as an alternative substrate to amniotic membrane.

Cells of renin lineage are adult pluripotent progenitors in experimental glomerular disease.

Modified vascular smooth muscle cells of the kidney afferent arterioles have recently been shown to serve as progenitors for glomerular epithelial cells in response to glomerular injury. To determine whether such cells of renin lineage (CoRL) serve as progenitors for other cells in kidney disease characterized by both glomerular and tubulointerstitial injury, permanent genetic cell fate mapping of adult CoRL using Ren1cCreER × Rs-tdTomato-R reporter mice was performed. TdTomato-labeled CoRL were almost completely restricted to the juxtaglomerular compartment in healthy kidneys. Following 2 wk of antibody-mediated focal segmental glomerulosclerosis (FSGS) or 16 wk of ⅚ nephrectomy-induced chronic kidney diseases, tdTomato-mapped CoRL were identified in both interstitial and glomerular compartments. In the interstitium, PDGFß receptor (R)-expressing cells significantly increased, and a portion of these expressed tdTomato. This was accompanied by a decrease in native pericyte number, but an increase in the number of tdTomato cells that coexpressed the pericyte markers PDGFß-R and NG2. These cells surrounded vessels and coexpressed the pericyte markers CD73 and CD146, but not the endothelial marker ERG. Within glomeruli of reporter mice with the ⅚ nephrectomy model, a subset of labeled CoRL migrated to the glomerular tuft and coexpressed podocin and synaptopodin. By contrast, labeled CoRL were not detected in glomerular or interstitial compartments following uninephrectomy. These observations indicate that in addition to supplying new adult podocytes to glomeruli, CoRL have the capacity to become new adult pericytes in the setting of interstitial disease. We conclude that CoRL have the potential to function as progenitors for multiple adult cell types in kidney disease.

Detection of abnormal extracellular matrix in the interstitium of regenerating renal tubules.

Stem/progenitor cells are promising candidates for the regeneration of parenchyma in acute and chronic renal failure. However, recent data exhibit that survival of stem/progenitor cells after implantation in diseased renal parenchyma is restricted. To elaborate basic parameters improving survival, cell seeding was simulated under advanced in vitro conditions. After isolation, renal stem/progenitor cells were mounted in a polyester interstitium for perfusion culture. During generation of tubules, chemically defined CO2 Independent Medium or Leibovitz's L-15 Medium was applied. Specimens were then fixed for transmission electron microscopy to analyze morphological features in generated tubules. Fixation in conventional glutaraldehyde (GA) solution shows development of tubules each exhibiting a polarized epithelium, an intact basal lamina and an inconspicuous interstitium. In contrast, special fixation of specimens in GA solution containing cupromeronic blue, ruthenium red or tannic acid unveils previously not visible extracellular matrix. Control experiments elucidate that a comparable extracellular matrix is not present in the interstitium of the matured kidney. Thus, generation of renal tubules in combination with advanced fixation of specimens for electron microscopy demonstrates that development of abnormal features in the newly developed interstitium has to be considered, when repair of renal parenchyma is performed by implantation of stem/progenitor cells.

Identification of adult stem cells in Schwalbe's line region of the primate eye.

PURPOSE: To identify stem cells in the chamber angle of the monkey eye by detection of 5-bromo-2'-deoxyuridine (BrdU) long-term retention. METHODS: Four cynomolgus monkeys were treated with BrdU via subcutaneous pumps for 4 weeks. The eyes of two animals were processed immediately thereafter (group 1) while in the other animals, BrdU treatment was discontinued for 4 weeks to allow identification of cells with long-term BrdU retention (group 2). The number of BrdU-positive nuclei was quantified, and the cells were characterized by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The number of BrdU-positive cells was higher at Schwalbe's line covering the peripheral end of Descemet's membrane than in Schlemm's canal (SC) endothelium, trabecular meshwork (TM), and scleral spur (SS). Labeling with BrdU in SC, TM, and SS was less intense and the number of labeled cells was smaller in group 2 than in group 1. In contrast, in cells of Schwalbe's line the intensity of BrdU staining and the number of BrdU-positive cells was similar when group 1 and 2 monkeys were compared with each other, indicating long-term BrdU retention. Cells that were BrdU-positive in Schwalbe's line region stained for the stem cell marker OCT4. Details of a stem cell niche in Schwalbe's line region were identified by TEM. CONCLUSIONS: We provide evidence for a niche in the Schwalbe's line region harboring cells with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a population of adult stem cells with the capability to compensate for the loss of TM and/or corneal endothelial cells.

Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.

Adult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.

Sensory-specific modulation of adult neurogenesis in sensory structures is associated with the type of stem cell present in the neurogenic niche of the zebrafish brain.

Teleost fishes retain populations of adult stem/progenitor cells within multiple primary sensory processing structures of the mature brain. Though it has commonly been thought that their ability to give rise to adult-born neurons is mainly associated with continuous growth throughout life, whether a relationship exists between the processing function of these structures and the addition of new neurons remains unexplored. We investigated the ultrastructural organisation and modality-specific neurogenic plasticity of niches located in chemosensory (olfactory bulb, vagal lobe) and visual processing (periventricular grey zone, torus longitudinalis) structures of the adult zebrafish (Danio rerio) brain. Transmission electron microscopy showed that the cytoarchitecture of sensory niches includes many of the same cellular morphologies described in forebrain niches. We demonstrate that cells with a radial-glial phenotype are present in chemosensory niches, while the niche of the caudal tectum contains putative neuroepithelial-like cells instead. This was supported by immunohistochemical evidence showing an absence of glial markers, including glial fibrillary acidic protein, glutamine synthetase, and S100ß in the tectum. By exposing animals to sensory assays we further illustrate that stem/progenitor cells and their neuronal progeny within sensory structures respond to modality-specific stimulation at distinct stages in the process of adult neurogenesis - chemosensory niches at the level of neuronal survival and visual niches in the size of the stem/progenitor population. Our data suggest that the adult brain has the capacity for sensory-specific modulation of adult neurogenesis and that this property may be associated with the type of stem cell present in the niche.

Mesothelial cells: a cellular surrogate for tissue engineering of corneal endothelium.

PURPOSE: To evaluate whether mouse adipose tissue mesothelial cells (ATMCs) share morphologic and biochemical characteristics with mouse corneal endothelial cells (CECs) and to evaluate their capacity to adhere to the decellularized basal membrane of human anterior lens capsules (HALCs) as a potential tissue-engineered surrogate for corneal endothelium replacement. METHODS: Adipose tissue mesothelial cells were isolated from the visceral adipose tissue of adult mice, and their expression of several corneal endothelium markers was determined with quantitative RT-PCR, immunofluorescence, and Western blotting. Adipose tissue mesothelial cells were cultured in a mesothelial retaining phenotype medium (MRPM) and further seeded and cultured on top of the decellularized basal membrane of HALCs. ATMC-HALC composites were evaluated by optical microscopy, immunofluorescence, and transmission electron microscopy. RESULTS: Mesothelial retaining phenotype medium-cultured ATMCs express the corneal endothelium markers COL4A2, COL8A2, SLC4A4, CAR2, sodium- and potassium-dependent adenosine triphosphatase (Na(+)/K(+)-ATPase), ß-catenin, zona occludens-1, and N-cadherin in a pattern similar to that in mouse CECs. Furthermore, ATMCs displayed strong adhesion capacity onto the basal membrane of HALCs and formed a confluent monolayer within 72 hours of culture in MRPM. Ultrastructural morphologic and marker characteristics displayed by ATMC monolayer on HALCs clearly indicated that ATMCs retained their original phenotype of squamous epithelial-like cells. CONCLUSIONS: Corneal endothelial cells and ATMCs share morphologic (structural) and marker (functional) similarities [corrected]. The ATMCs adhered and formed structures mimicking focal adhesion complexes with the HALC basal membrane. Monolayer structure and achieved density of ATMCs support the proposal to use adult human mesothelial cells (MCs) as a possible surrogate for damaged corneal endothelium.

Colony-forming progenitor cells in the postnatal mouse liver and pancreas give rise to morphologically distinct insulin-expressing colonies in 3D cultures.

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed "Dark" colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133⁺CD49f(low)CD107b(low) phenotype, while pancreatic CFU-Dark are CD133⁻. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth.

Kidney regeneration by non-platelet RNA-containing particle-derived cells.

We found a group of non-platelet RNA-containing particles (NPRCPs) in human umbilical cord blood. These particles can aggregate, fuse and become non-nucleated cells when cocultured with nucleated cells in vitro. The non-nucleated cells further differentiate into nucleated cells expressing octamer binding transcription factor 4 (OCT4). The NPRCPs are approximately 1-5 µm in diameter, have a thin bilayer membrane, contain short RNAs and microRNAs and express OCT4, sex-determining region Y 2 (SOX2) and DEAD box polypeptide 4 (DDX4). To confirm the function of NPRCPs in vivo, we examined the effects of tail vein-injected green fluorescent protein (GFP)-labelled NPRCPs on mouse kidneys damaged by prior ischaemia and reperfusion from Day 1 to Week 6. Within 1 day of injection of NPRCPs, immunofluorescence and immunohistochemistry revealed a large number of extravasated NPRCPs in the renal calyces, damaged glomeruli and duct tubules. During the course of regeneration, NPRCPs fused into large, non-nucleated cellular structures that further became large nucleated cells to regenerate multicellular kidney tubules. In addition, many NPRCPs became tiny nucleated cellular structures that further differentiated into interstitial cells in connective tissue. The extravasated NPRCPs also arranged themselves into non-cell glomerular structures before further regenerating into nucleated cells of the glomerulus. In conclusion, the results demonstrate that, via different patterns of differentiation, NPRCP-derived cells can regenerate mouse kidney tissue damaged by ischaemia.

The effects of Ca2SiO4-Ca3(PO4)2 ceramics on adult human mesenchymal stem cell viability, adhesion, proliferation, differentiation and function.

Bioceramic samples with osteogenic properties, suitable for use in the regeneration of hard tissue, were synthesized. The materials consisting of α-tricalcium phosphate (αTCP) and also αTCP doped with either 1.5 wt.% or 3.0 wt.% of dicalcium silicate (C2S) in the system Dicalcium Silicate-Tricalcium Phosphate (C2S-TCP) were obtained by solid state reaction. All materials were composed of a single phase, αTCP in the case of a pure material, or solid solution of C2S in αTCP (αTCPss) for the doped αTCP. Viability, proliferation and in vitro osteoinductive capacity were investigated by seeding, adult mesenchymal stem cells of human origin (ahMSCs) which were CD73(+), CD90(+), CD105(+), CD34(-) and CD45(-) onto the 3 substrates for 30 days. Results show a non-cytotoxic effect after applying an indirect apoptosis test (Annexin V/7-AAD staining), so ahMSCs adhered, spread, proliferated and produced extracellular matrix (Heparan-sulfate proteoglycan (HS) and osteopontin (OP)) on all the ceramics studied. Finally, the cells lost the cluster differentiation marker expression CD73, CD90 y CD105 characteristic of ahMSCs and they showed an osteoblastic phenotype (Alkaline phosphatase activity (ALP), Osteocalcin production (OC), Collagen type I expression (Col-I), and production of mineralization nodules on the extracellular matrix). These observations were more evident in the αTCP ceramic doped with 1.5 wt.% C2S, indicating osteoblastic differentiation as a result of the increased concentration of solid solution of C2S in αTCP (αTCPss). Overall, these results suggest that the ceramics studied are cytocompatible and they are able to induce osteoblastic differentiation of undifferentiated ahMSCs.