Spatiotemporal extracellular matrix modeling for in situ cell niche studies.

Extracellular matrix (ECM) components govern a range of cell functions, such as migration, proliferation, maintenance of stemness, and differentiation. Cell niches that harbor stem-/progenitor cells, with matching ECM, have been shown in a range of organs, although their presence in the heart is still under debate. Determining niches depends on a range of in vitro and in vivo models and techniques, where animal models are powerful tools for studying cell-ECM dynamics; however, they are costly and time-consuming to use. In vitro models based on recombinant ECM proteins lack the complexity of the in vivo ECM. To address these issues, we present the spatiotemporal extracellular matrix model for studies of cell-ECM dynamics, such as cell niches. This model combines gentle decellularization and sectioning of cardiac tissue, allowing retention of a complex ECM, with recellularization and subsequent image processing using image stitching, segmentation, automatic binning, and generation of cluster maps. We have thereby developed an in situ representation of the cardiac ECM that is useful for assessment of repopulation dynamics and to study the effect of local ECM composition on phenotype preservation of reseeded mesenchymal progenitor cells. This model provides a platform for studies of organ-specific cell-ECM dynamics and identification of potential cell niches.

Vagal-α7nAChR signaling promotes lung stem cells regeneration via fibroblast growth factor 10 during lung injury repair.

BACKGROUND: Proliferation and transdifferentiation of lung stem cells (LSCs) could promote lung injury repair. The distal airways of the lung are innervated by the vagus nerve. Vagal-alpha7 nicotinic acetylcholine receptor (α7nAChR) signaling plays a key role in regulating lung infection and inflammation; however, whether this pathway could regulate LSCs remains unknown. METHODS: LSCs (Sca1+CD45-CD31- cells) were isolated and characterized according to a previously published protocol. α7nAChR knockout mice and wild-type littermates were intratracheally challenged with lipopolysaccharide (LPS) to induce lung injury. A cervical vagotomy was performed to study the regulatory effect of the vagus nerve on LSCs-mediated lung repair. α7nAChR agonist or fibroblast growth factor 10 (FGF10) was intratracheally delivered to mice. A single-cell suspension of lung cells was analyzed by flow cytometry. Lung tissues were collected for histology, quantitative real-time polymerase chain reaction (RT-PCR), and immunohistochemistry. RESULTS: We found that LSCs maintained multilineage differentiation ability and transdifferentiated into alveolar epithelial type II cells (AEC2) following FGF10 stimulation in vitro. Vagotomy or α7nAChR deficiency reduced lung Ki67+ LSCs expansion and hampered the resolution of LPS-induced lung injury. Vagotomy or α7nAChR deficiency decreased lung FGF10 expression and the number of AEC2. The α7nAChR agonist-GTS-21 reversed the reduction of FGF10 expression in the lungs, as well as the number of Ki67+ cells, LSCs, Ki67+ LSCs, and AEC2 in LPS-challenged vagotomized mice. Supplementation with FGF10 counteracted the loss of Ki67+ LSCs and AEC2 in LPS-challenged α7nAChR knockout mice. CONCLUSIONS: The vagus nerve deploys α7nAChR to enhance LSCs proliferation and transdifferentiation and promote lung repair in an FGF10-dependent manner during LPS-induced lung injury.

A comprehensive characterisation of large-scale expanded human bone marrow and umbilical cord mesenchymal stem cells.

BACKGROUND: The manufacture of mesenchymal stem/stromal cells (MSCs) for clinical use needs to be cost effective, safe and scaled up. Current methods of expansion on tissue culture plastic are labour-intensive and involve several 'open' procedures. We have used the closed Quantum® hollow fibre bioreactor to expand four cultures each of MSCs derived from bone marrow (BM) and, for the first time, umbilical cords (UCs) and assessed extensive characterisation profiles for each, compared to parallel cultures grown on tissue culture plastic. METHODS: Bone marrow aspirate was directly loaded into the Quantum®, and cells were harvested and characterised at passage (P) 0. Bone marrow cells were re-seeded into the Quantum®, harvested and further characterised at P1. UC-MSCs were isolated enzymatically and cultured once on tissue culture plastic, before loading cells into the Quantum®, harvesting and characterising at P1. Quantum®-derived cultures were phenotyped in terms of immunoprofile, tri-lineage differentiation, response to inflammatory stimulus and telomere length, as were parallel cultures expanded on tissue culture plastic. RESULTS: Bone marrow cell harvests from the Quantum® were 23.1 ± 16.2 × 106 in 14 ± 2 days (P0) and 131 ± 84 × 106 BM-MSCs in 13 ± 1 days (P1), whereas UC-MSC harvests from the Quantum® were 168 ± 52 × 106 UC-MSCs after 7 ± 2 days (P1). Quantum®- and tissue culture plastic-expanded cultures at P1 adhered to criteria for MSCs in terms of cell surface markers, multipotency and plastic adherence, whereas the integrins, CD29, CD49c and CD51/61, were found to be elevated on Quantum®-expanded BM-MSCs. Rapid culture expansion in the Quantum® did not cause shortened telomeres when compared to cultures on tissue culture plastic. Immunomodulatory gene expression was variable between donors but showed that all MSCs upregulated indoleamine 2, 3-dioxygenase (IDO). CONCLUSIONS: The results presented here demonstrate that the Quantum® can be used to expand large numbers of MSCs from bone marrow and umbilical cord tissues for next-generation large-scale manufacturing, without impacting on many of the properties that are characteristic of MSCs or potentially therapeutic. Using the Quantum®, we can obtain multiple MSC doses from a single manufacturing run to treat many patients. Together, our findings support the development of cheaper cell-based treatments.

Stable overexpression of p130/E2F4 affects the multipotential abilities of bone-marrow-derived mesenchymal stem cells.

Bone-marrow-derived mesenchymal stem cells (MSCs) have great potential in transplantation medicine due to their multiple advantages. However, the controlled differentiation of MSCs is one of the key aspects of effective clinical transplantation. Growing evidence suggests that the cell cycle plays an important role in regulating differentiation, while p130 and E2F4 are key to cell cycle checkpoints. The aim of the study is to evaluate the effects and mechanism of p130/E2F4 on the multidifferentiation of MSCs. Our data showed that the transduction efficiencies of p130 or E2F4 mediated by lentiviral vectors were 80.3%-84.4%. p130 and E2F4 mRNA expression was significantly higher in MSC-p130 and MSC-E2F4 cells than in MSC normal control (NC) cells. Similar results were also observed for p130 and E2F4 protein expression. After osteogenic or adipogenic differentiation, the G1 phase was significantly delayed in the MSC-p130 and MSC-E2F4 groups compared with that in the MSC-NC group. However, the G1 phase in the MSC-p130 and MSC-E2F4 groups did the opposite after chondrogenic differentiation. Moreover, overexpressing p130 or E2F4 significantly improved osteogenic differentiation while inhibiting adipogenic and chondrogenic differentiation of mouse MSCs (mMSCs). Moreover, overexpressing p130 or E2F4 significantly improved migration but not proliferation of mMSCs. Our data suggest that cell cycle regulation may be involved in p130/E2F4-mediated changes in the multipotential abilities of bone-marrow-derived mMSCs.

Alpha-Ketoglutarate Curbs Differentiation and Induces Cell Death in Mesenchymal Stromal Precursors with Mitochondrial Dysfunction.

Increased concentrations of reactive oxygen species (ROS) originating from dysfunctional mitochondria contribute to diverse aging-related degenerative disorders. But so far little is known about the impact of distinct ROS on metabolism and fate of stromal precursor cells. Here, we demonstrate that an increase in superoxide anion radicals due to superoxide dismutase 2 (Sod2) deficiency in stromal precursor cells suppress osteogenic and adipogenic differentiation through fundamental changes in the global metabolite landscape. Our data identify impairment of the pyruvate and l-glutamine metabolism causing toxic accumulation of alpha-ketoglutarate in the Sod2-deficient and intrinsically aged stromal precursor cells as a major cause for their reduced lineage differentiation. Alpha-ketoglutarate accumulation led to enhanced nucleocytoplasmic vacuolation and chromatin condensation-mediated cell death in Sod2-deficient stromal precursor cells as a consequence of DNA damage, Hif-1α instability, and reduced histone H3 (Lys27) acetylation. These findings hold promise for prevention and treatment of mitochondrial disorders commonly associated with aged individuals. Stem Cells 2017;35:1704-1718.

[Research progress of miRNA regulation in differentiation of adipose-derived stem cells].

Objective: To review the research progress of miRNA regulation in the differentiation of adipose-derived stem cells (ADSCs). Methods: The recent literature associated with miRNAs and differentiation of ADSCs was reviewed. The regulatory mechanism was analyzed in detail and summarized. Results: The results indicate that the expression of miRNAs changes during differentiation of ADSCs. In addition, miRNAs regulate the differentiation of ADSCs into adipocytes, osteoblasts, chondrocytes, neurons, and hepatocytes by regulating the signaling pathways involved in cell differentiation. Conclusion: Through controlling the differentiation of ADSCs by miRNAs, the suitable seed cell for tissue engineering can be established. The review will provide a theoretical basis for molecular targeted therapy and stem cell therapy in clinic.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Differentiation of hepatocytes from induced pluripotent stem cells derived from human hair follicle mesenchymal stem cells.

Due to the limitations of organ donors and immune rejection in severe liver diseases, stem cell-based therapy presents a promising application for tissue repair and regeneration. As a novel cell source, mesenchymal stem cells separated from human hair follicles (HF-MSCs) are convenient to obtain and have no age limit. To date, the differentiation of HF-MSCs into hepatocytes has not been reported. In this study, we explored whether HF-MSCs and HF-MSC-derived-induced pluripotent stem cells (HF-iPS) could differentiate into hepatocytes in vitro. Flow cytometry, Oil Red O stain and Alizarin Red stain were used to identify the characteristics of HF-MSCs. The expression of liver-specific gene was detected by immunofluorescence and Quantitative Polymerase Chain Reaction. Periodic Acid-Schiff stain, Indocyanine Green stain and Low-Density Lipoprotein stain were performed to evaluate the functions of induced hepatocyte-like cells (HLCs). HF-MSCs were unable to differentiate into HLCs using previously reported procedures for MSCs from other tissues. However, HF-iPS efficiently induced the generation of HLCs that expressed hepatocyte markers and drug metabolism-related genes. HF-iPS can be used as novel and alternative cellular tools for inducing hepatocytes in vitro, simultaneously benefiting from utilizing HF-MSCs as a noninvasive and convenient cell source for reprogramming.

Epidermal stem cells and skin tissue engineering in hair follicle regeneration.

The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients' psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This field is attractive not only to academic researchers but also to the companies that own almost half of the patents in this field.

Dermal papilla cells improve the wound healing process and generate hair bud-like structures in grafted skin substitutes using hair follicle stem cells.

Tissue-engineered skin represents a useful strategy for the treatment of deep skin injuries and might contribute to the understanding of skin regeneration. The use of dermal papilla cells (DPCs) as a dermal component in a permanent composite skin with human hair follicle stem cells (HFSCs) was evaluated by studying the tissue-engineered skin architecture, stem cell persistence, hair regeneration, and graft-take in nude mice. A porcine acellular dermal matrix was seeded with HFSCs alone and with HFSCs plus human DPCs or dermal fibroblasts (DFs). In vitro, the presence of DPCs induced a more regular and multilayered stratified epidermis with more basal p63-positive cells and invaginations. The DPC-containing constructs more accurately mimicked the skin architecture by properly stratifying the differentiating HFSCs and developing a well-ordered epithelia that contributed to more closely recapitulate an artificial human skin. This acellular dermal matrix previously repopulated in vitro with HFSCs and DFs or DPCs as the dermal component was grafted in nude mice. The presence of DPCs in the composite substitute not only favored early neovascularization, good assimilation and remodeling after grafting but also contributed to the neovascular network maturation, which might reduce the inflammation process, resulting in a better healing process, with less scarring and wound contraction. Interestingly, only DPC-containing constructs showed embryonic hair bud-like structures with cells of human origin, presence of precursor epithelial cells, and expression of a hair differentiation marker. Although preliminary, these findings have demonstrated the importance of the presence of DPCs for proper skin repair.

sdf1 Expression reveals a source of perivascular-derived mesenchymal stem cells in zebrafish.

There is accumulating evidence that mesenchymal stem cells (MSCs) have their origin as perivascular cells (PVCs) in vivo, but precisely identifying them has been a challenge, as they have no single definitive marker and are rare. We have developed a fluorescent transgenic vertebrate model in which PVC can be visualized in vivo based upon sdf1 expression in the zebrafish. Prospective isolation and culture of sdf1(DsRed) PVC demonstrated properties consistent with MSC including prototypical cell surface marker expression; mesodermal differentiation into adipogenic, osteogenic, and chondrogenic lineages; and the ability to support hematopoietic cells. Global proteomic studies performed by two-dimensional liquid chromatography and tandem mass spectrometry revealed a high degree of similarity to human MSC (hMSC) and discovery of novel markers (CD99, CD151, and MYOF) that were previously unknown to be expressed by hMSC. Dynamic in vivo imaging during fin regeneration showed that PVC may arise from undifferentiated mesenchyme providing evidence of a PVC-MSC relationship. This is the first model, established in zebrafish, in which MSC can be visualized in vivo and will allow us to better understand their function in a native environment.

Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients.

Transplantation of CD15-enriched murine neural stem cells increases total engraftment and shifts differentiation toward the oligodendrocyte lineage.

Neural stem cell (NSC) transplantation is a promising therapeutic approach for neurological diseases. However, only a limited number of cells can be transplanted into the brain, resulting in relatively low levels of engraftment. This study investigated the potential of using a cell surface marker to enrich a primary NSC population to increase stable engraftment in the recipient brain. NSCs were enriched from the neonatal mouse forebrain using anti-CD15 (Lewis X antigen, or SSEA-1) in a "gentle" fluorescence-activated cell sorting protocol, which yielded >98% CD15-positive cells. The CD15-positive cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, after withdrawal of growth factors, demonstrating multipotentiality. CD15-positive cells were expanded in vitro and injected bilaterally into the ventricles of neonatal mice. Cells from enriched and unenriched donor populations were found throughout the neuraxis, in both neurogenic and non-neurogenic regions. Total engraftment was similar at 7 days postinjection, but by 28 days postinjection, after brain organogenesis was complete, the survival of donor cells was significantly increased in CD15-enriched grafts over the unenriched cell grafts. The engrafted cells were heterogeneous in morphology and differentiated into all three neural lineages. Furthermore, in the CD15-enriched grafts, there was a significant shift toward differentiation into oligodendrocytes. This strategy may allow better delivery of therapeutic cells to the developing central nervous system and may be particularly useful for treating diseases involving white matter lesions.