Derived Stem Cells Research Articles

BMP-2/TGF-β1 gene insertion into ligament-derived stem cells sheet promotes tendon-bone healing in a mouse.

Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) reportedly induce the osteogenic and tenogenic differentiation of anterior cruciate ligament (ACL)-derived stem cells (LDSCs), respectively. However, few studies have investigated the effect of BMP-2/TGF-β1 on the differentiation of LDSC. We developed a BMP-2/TGF-β1 gene insertion into an LDSC cell sheet that promotes tendon-bone healing in a mouse ACL reconstruction (ACLR) model. CD34+ LDSCs were isolated from human ACL stump tissues, virally transduced to express BMP-2 or TGF-β1, and then embedded within cell sheets. All mice underwent ACLR using an autograft wrapped with a cell sheet and were randomly divided into three groups: BMP-2-, TGF-β1-, and BMP-2/TGF-β1-transduced. At 4 and 8weeks, tendon-bone healing was evaluated by micro-CT, biomechanical test, and histological analysis. BMP-2 and TGF-β1 promoted the osteogenic and tenogenic differentiation of LDSC in vitro. BMP-2/TGF-β1-transduced LDSC sheet application contributed to early improvement in mean failure load and graft stiffness, accelerated maturation of the tendon-bone junction, and inhibited bone tunnel widening. Furthermore, reduced M1 macrophage infiltration and a higher M2 macrophage percentage were observed in the BMP-2/TGF-β1-transduced LDSC group. This work demonstrated that BMP-2 and TGF-β1 promoted CD34+ LDSCs osteogenic and tenogenic differentiation in vitro and in vivo, which accelerated the tendon-bone healing after ACLR using autografts wrapped with cell sheets in a mouse model.

Combining single-cell transcriptomics and CellTagging to identify differentiation trajectories of human adipose-derived mesenchymal stem cells

BackgroundMesenchymal stromal cells (MSCs) have attracted great attention in the application of cell-based therapy because of their pluripotent differentiation and immunomodulatory ability. Due to the limited number of MSCs isolated from donor tissues, a large number of MSCs need to be expanded in a traditional two-dimensional cell culture device to obtain a sufficient therapeutic amount. However, long-term cultivation of MSCs in vitro has been proven to reduce their differentiation potential and change their immunomodulatory characteristics. We aimed to explore the cellular heterogeneity and differentiation potential of different MSCs expanded in vitro and reconstruct the complex cloning track of cells in the process of differentiation.MethodsSingle cell transcriptome sequencing was combined with ‘CellTagging’, which is a composite barcode indexing method that can capture the cloning history and cell identity in parallel to track the differentiation process of the same cell over time.ResultsThrough the single-cell transcriptome and CellTagging, we found that the heterogeneity of human adipose tissue derived stem cells (hADSCs) in the early stage of culture was very limited. With the passage, the cells spontaneously differentiated during the process of division and proliferation, and the heterogeneity of the cells increased. By tracing the differentiation track of cells, we found most cells have the potential for multidirectional differentiation, while a few cells have the potential for unidirectional differentiation. One subpopulation of hADSCs with the specific osteoblast differentiation potential was traced from the early stage to the late stage, which indicates that the differentiation trajectories of the cells are determined in the early stages of lineage transformation. Further, considering that all genes related to osteogenic differentiation have not yet been determined, we identified that there are some genes that are highly expressed specifically in the hADSC subsets that can successfully differentiate into osteoblasts, such as Serpin Family E Member 2 (SERPINE2), Secreted Frizzled Related Protein 1 (SFRP1), Keratin 7 (KRT7), Peptidase Inhibitor 16 (PI16), and Carboxypeptidase E (CPE), which may be key regulatory genes for osteogenic induction, and finally proved that the SERPINE2 gene can promote the osteogenic process.ConclusionThe results of this study contribute toward the exploration of the heterogeneity of hADSCs and improving our understanding of the influence of heterogeneity on the differentiation potential of cells. Through this study, we found that the SERPINE2 gene plays a decisive role in the osteogenic differentiation of hADSCs, which lays a foundation for establishing a more novel and complete induction system.

An animal model study of osteochondral defect repair by human adipose stem cells and pomegranate fruit hydroalchoholic extract.

Articular cartilage damages do not repair spontaneously. Tissue engineering is a promising approach to repair cartilage damage. Transforming growth factor-beta (TGF-β) members are the known induction factors in chondrogenic differentiation. However, hypertrophy of the chondrocytes resulting from mesenchymal stem cells (MSCs) induction by TGF-β is inevitable. Pomegranate fruit contains many ingredients which are useful in ensuring the health of organs. This study was designed to investigate the Pomegranate Fruit hydroalchoholic Extract (PFE) capability in human adipose derived stem cells (hASCs) differentiation into the chondrocytes on fibrin scaffold. Pomegranate fruit hydroalchoholic extract (PFE) was prepared. hASCs were isolated, expanded, labeled, and seeded on the fibrin scaffold. The constructs were divided into three groups including TGF-β3, PFE, and control. The constructs were induced for 14 days, then, the MTT assay, Real-Time Polymerase Chain Reaction (PCR), and histochemistry assessments were run, and finally, the constructs were transplanted into the knee defect of rats. The gross and histological assessments of the transplants were done after 8 weeks. The viability rate, COL2A1, Aggrecan (ACAN) and COL10A1 genes expression levels, and histological criterion of the PFE samples were significantly higher than that of the control. The macroscopic grades and histological results of the PFE samples were close to that of the TGF-β3. The number of positive cells for COLІI protein were higher significantly in the PFE group than the control. PFE was effective in the chondrogenic induction of hASCs. Further studies are needed to find out the events of the chondrogenic induction using PFE.

ALK-5 Inhibitors for Efficient Derivation of Mesenchymal Stem Cells from Human Embryonic Stem Cells.

Objectives: Successful tissue regeneration requires a clinically viable source of mesenchymal stem cells (MSCs). We explored activin receptor-like kinase (ALK)-5 inhibitors to rapidly derive an MSC-like phenotype with high cartilage forming capacity from a xeno-free human embryonic cell line. Methods: Embryonic stem cell (ESC) lines (H9 and HADC100) were treated with the ALK-5 inhibitor SB431542; HADC100 cells were additionally treated with ALK-5 inhibitors SB525334 or GW788388. Cells were then seeded upon human fibronectin in the presence of fibroblast growth factor 2 (FGF2) in a serum-free medium. Flow cytometry was used to assess MSC markers (positive for CD73, CD90, and CD105; negative for CD34 and CD45). Differentiation status was assessed through quantitative polymerase chain reaction. Cartilage forming capacity was determined in high-density pellet cultures, in fibrin gels containing extracellular matrix (fibrin-ECM), and after implantation in ex vivo human osteoarthritic cartilage. Gene expression, histology, and immunostaining were used to assess cartilage phenotype, tissue regeneration, and integration. Results: Exposure to all three ALK-5 inhibitors lead to expression of mesodermal gene markers and differentiation into MSC-like cells (embryonic stem cell-derived mesenchymal stem cells [ES-MSCs]) based on surface marker expression. ES-MSC in pellet cultures or in fibrin-ECM gels expressed high levels of chondrogenic genes: COL2A1, ACAN, and COMP; and low levels of COL1A1 and RUNX2. Cell pellets or fibrin constructs implanted into ex vivo human osteoarthritic cartilage defects produced GAG-rich (safranin O positive) and collagen type II-positive neocartilage tissues that integrated well with native diseased tissue. Conclusions: We developed a protocol for rapid differentiation of xeno-free ESC into MSC-like cells with high cartilage forming capacity with potential for clinical applications. Impact statement Osteoarthritis (OA) is a common disease resulting in significant disability and no approved disease modifying treatment (other than total joint replacement). Embryonic stem cell-derived cell therapy has the potential to benefit patients with cartilage lesions leading to OA and may prevent or delay the need for total joint replacement.

Fat grafting: an adjunct in management of chronic leg ulcers

Chronic leg ulcers (CLU’s) are one of the most challenging medical conditions, mostly resistant to conventional therapy. Several treatment modalities are present but none have become standard of care. Autologous fat contains adipose derived stem cells (ADSC’s) which is believed to have abundant growth factors (GF’s) required for its regenerative and reparative properties. Our observational study was conducted to see the feasibility and efficacy of fat grafting in management of CLUs with complete wound healing as final outcome. 14 patients with CLUs (any etiology, >3 months and multiple ulcers were counted as individual cases if >3 cm apart) were included with coagulopathies, uncontrolled diabetes mellitus (Hba1c>9 mmol/mol) being the exclusions. Fat was harvested using standard Coleman’s technique. Study was conducted from December 2015 to December 2017 in tertiary care hospital and patients were followed up for next 24 months. All cases were evaluated clinically. Average age of wounds before intervention was 33.34 months, average wound size 16.46 cm2 and 4.9 cc of average fat was transferred underneath the ulcer. 11 out of 14 wounds healed completely within average 27.4 days and final outcome was not achieved in 3. Pain assessed using visual analogue scale (VAS) in immediate post operative period till 48 hours (telephonically). Patients were evaluated at 1 week, 1 month, 3 months and 6 months after procedure for pattern of wound healing and related complications. Fat grafting is easy, safe, effective and a minimally invasive OPD procedure with short recovery time for managing CLUs.

Derivation of Limbal Stem Cells from Human Adult Mesenchymal Stem Cells for the Treatment of Limbal Stem Cell Deficiency.

Approximately 10 million individuals have blindness due to limbal stem cell (LSCs) deficiency, one of the most challenging problems in ophthalmology. To replenish the LSC pool, an autologous extraocular cell source is appropriate, thereby avoiding the risk of immune rejection, the need for immunosuppression and the risk of damaging the contralateral eye. In recent years, adipose-derived mesenchymal stem cells (ADSCs) have been a key element in ocular regenerative medicine. In this study, we developed a protocol for deriving human LSCs from ADSCs compatible with the standard carrier human amniotic membrane, helping provide a stem cell pool capable of maintaining proper corneal epithelial homeostasis. The best protocol included an ectodermal induction step by culturing ADSCs with media containing fetal bovine serum, transforming growth factor-β inhibitor SB-505124, Wnt inhibitor IWP-2 and FGF2 for 7 days, followed by an LSC induction step of culture in modified supplemental hormonal epithelial medium supplemented with pigment epithelium-derived factor and keratinocyte growth factor for 10 additional days. The optimal differentiation efficiency was achieved when cells were cultured in this manner over vitronectin coating, resulting in up to 50% double-positive αp63/BMI-1 cells. The results of this project will benefit patients with LSC deficiency, aiding the restoration of vision.

Efficient generation of ETX embryoids that recapitulate the entire window of murine egg cylinder development.

The murine embryonic-trophoblast-extra-embryonic endoderm (ETX) model is an integrated stem cell-based model to study early postimplantation development. It is based on the self-assembly potential of embryonic, trophoblast, and hypoblast/primitive/visceral endoderm-type stem cell lines (ESC, TSC, and XEN, respectively) to arrange into postimplantation egg cylinder-like embryoids. Here, we provide an optimized method for reliable and efficient generation of ETX embryoids that develop into late gastrulation in static culture conditions. It is based on transgenic Gata6-overproducing ESCs and modified assembly and culture conditions. Using this method, up to 43% of assembled ETX embryoids exhibited a correct spatial distribution of the three stem cell derivatives at day 4 of culture. Of those, 40% progressed into ETX embryoids that both transcriptionally and morphologically faithfully mimicked in vivo postimplantation mouse development between E5.5 and E7.5. The ETX model system offers the opportunity to study the murine postimplantation egg cylinder stages and could serve as a source of various cell lineage precursors.

Metformin promotes angiogenesis by enhancing VEGFa secretion by adipose-derived stem cells via the autophagy pathway.

Human adipose tissue-derived stem cell (ADSC) derivatives are cell-free, with low immunogenicity and no potential tumourigenicity, making them ideal for aiding wound healing. However, variable quality has impeded their clinical application. Metformin (MET) is a 5' adenosine monophosphate-activated protein kinase activator associated with autophagic activation. In this study, we assessed the potential applicability and underlying mechanisms of MET-treated ADSC derivatives in enhancing angiogenesis. We employed various scientific techniques to evaluate the influence of MET on ADSC, assess angiogenesis and autophagy in MET-treated ADSC in vitro, and examine whether MET-treated ADSC increase angiogenesis. We found that low MET concentrations exerted no appreciable effect on ADSC proliferation. However, MET was observed to enhance the angiogenic capacity and autophagy of ADSC. MET-induced autophagy was associated with increased vascular endothelial growth factor A production and release, which contributed to promoting the therapeutic efficacy of ADSC. In vivo experiments confirmed that in contrast to untreated ADSC, MET-treated ADSC promoted angiogenesis. Our findings thus indicate that the application of MET-treated ADSC would be an effective approach to accelerate wound healing by promoting angiogenesis at wound sites.

Stem Cells Applications in Neurodegenerative Diseases

Neurodegenerative diseases are a kind of diseases caused by progressive loss of neuronal structure and function and glial cell homeostasis imbalance, there are many kinds of neurodegenerative diseases include Alzheimer's disease (AD), Parkinson's disease (PD); Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). So far, due to the lack of ideal treatment methods, it seriously threats to human health especially the elder population. Recently, with the rapid development of regenerative medicine, stem cells rely on their advantages including self-renewing capability, low immunogenicity, migration and homing capabilities, and stem cell derivatives including stem cells derived extracellular vesicles and stem cell-derived organoids, it provides unlimited application possibilities for the treatment of neurodegenerative diseases. In this review, we will summarize the recent research progress on the preclinical and clinical applications of stem cells in neurodegenerative diseases, hope that the reviews may provide some useful clues for researchers.

Different methods of bone marrow harvesting influence cell characteristics and purity, affecting clinical outcomes

BackgroundBone marrow (BM)-derived stem cells were implanted to induce angiogenesis in patients with no-option critical limb-threatening ischemia. Considering the potential for this therapy, conflicting results related to BM harvesting methods have been reported that could affect stem cell concentrations and quality. MethodsA total of 75 patients with no-option critical limb-threatening ischemia were treated with BM implantation. For 58 patients, BM was harvested using a BM aspirate concentrate system (Harvest Technologies; group HT) with a standard aspiration needle, followed by an automated centrifugation process, to produce BM aspirate concentrate. For 17 patients, BM was harvested using the Marrow Cellution system (Aspire Medical Innovation; group MC). CD34+ cells/mL, CD117+ cells/mL, CD133+ cells/mL, CD309+ cells/mL, hematocrit, and BM purity were compared between the two BM preparations. ResultsThe retrospective analysis of a subset group after adjustment for age shows that the quality of BM obtained using the Marrow Cellution system is better, in terms of purity, than the classic harvesting method before centrifugation. Harvested BM before centrifugation is characterized by a higher percentage of CD133+ cells compared with BM after centrifugation. In contrast, the MC aspirate had a larger amount of very small embryonic-like cells, as indicated by the higher percentage of CD133+, CD34+, and CD45− cells. These differences translated into an increased occurrence of leg amputations in group HT than in group MC and an increase in transcutaneous oxygen pressure in patients treated with BM aspirated using MC. ConclusionsBM manipulation, such as centrifugation, affects the quality and number of stem cells, with detrimental consequences on clinical outcomes, as reflected by the different amputation rates between the two groups.

Stem Cells as Nuclear Donors for Mammalian Cloning.

Mammals are routinely cloned by introducing somatic nuclei into enucleated oocytes. Cloning contributes to propagating desired animals, to germplasm conservation efforts, among other applications. A challenge to more broader use of this technology is the relatively low cloning efficiency, which inversely correlates with donor cell differentiation status. Emerging evidence suggests that adult multipotent stem cells improve cloning efficiency, while the greater potential of embryonic stem cells for cloning remains restricted to the mouse. The derivation of pluripotent or totipotent stem cells from livestock and wild species and their association with modulators of epigenetic marks in donor cells should increase cloning efficiency.

Derivation of Bovine Primed Embryonic Stem Cells from Somatic Cell Nuclear Transfer Embryos.

Derivation of bovine embryonic stem cells from somatic cell nuclear transfer embryos enables the derivation of genetically matched pluripotent stem cell lines to valuable and well-characterized animals. In this chapter, we describe a step-by-step procedure for deriving bovine embryonic stem cells from whole blastocysts produced by somatic cell nuclear transfer. This simple method requires minimal manipulation of blastocyst-stage embryos, relies on commercially available reagents, supports trypsin passaging, and allows the generation of stable primed pluripotent stem cell lines in 3-4weeks.

Derivation and Primordial Germ Cell Induction of Intermediate Pluripotent Stem Cells.

Dynamic pluripotent stem cell (PSC) states are in vitro adaptations of the pluripotency continuum in vivo. Previous studies have generated a number of PSCs with distinct properties. By modulating the FGF, TGF-β, and WNT pathways, we have derived intermediate PSCs (FTW-PSCs) that are permissive for direct primordial germ cell-like cell (PGC-LC) induction in vitro. Here, we describe the method for derivation and maintenance of mouse and human FTW-PSCs, as well as PGC-LC induction from FTW-PSCs.

In Vivo Real-Time Quantum Dots Imaging to Track Transplanted Adipose Stem Cells in Different Inflammatory States of Acute Liver Failure Mice

Stem cell therapy plays an important role in regenerative therapy; however, there is little information on the in vivo dynamics of transplanted stem cells and the influence of the inflammation of affected tissues or organs on these dynamics. In this study, we revealed real-time dynamics of transplanted adipose tissue–derived stem cells (ASCs) and the influence of the inflammatory states on these dynamics in acute liver failure mice. Quantum dots (QDs) labeling did not affect the cytokine profile of ASCs, and intravenously transplanted ASCs labeled with QDs could be detected in real time with high efficiency without laparotomy. Until 30 min after ASC transplantation, no marked differences in the behavior or accumulation of transplanted ASCs in the liver were observed among the three groups with different degrees of liver damage (normal, weak, and strong). However, significant differences in the engraftment rate of transplanted ASCs in the liver were observed among the three groups from 4 h after transplantation. The engraftment rate was inversely correlated with the extent of the liver damage. These data suggested that QDs are useful for in vivo real-time imaging of transplanted cells, and the inflammatory state of tissues or organs may affect the engraftment rate of transplanted cells.

Isogeometric simulation of a derived stem cell engineered ventricle

Isogeometric simulation of a derived stem cell engineered ventricle

Human Adipose Derived Stem Cells in Female Infertility Treatment

Human Adipose Derived Stem Cells in Female Infertility Treatment

Exosomes From Adipose-Derived Stem Cells Suppress the Progression of Chronic Endometritis

Chronic endometritis (CE) is closely linked to the reproductive failure. Exosome (Exo)-based therapy is proposed as an encouraging strategy in inflammation-related disorders; however, little work has been devoted to its usage in CE therapy. An in vitro CE was established by administration of lipopolysaccharide (LPS) in human endometrial stromal cells (HESCs). The cell proliferation, cell apoptosis, and inflammatory cytokine assays were performed in vitro, and the efficacy of Exos derived from adipose tissue–derived stem cells (ADSCs) was evaluated in a mouse model of CE. We found that Exos isolated from ADSCs could be taken up by HESCs. Exos elevated the proliferation and inhibited apoptosis in LPS-treated HESCs. Administration of Exos to HESCs suppressed the content of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Moreover, Exos exposure repressed the inflammation stimulated by LPS in vivo. Mechanistically, we demonstrated that Exos exerted their ant-inflammatory effect via miR-21/TLR4/NF-kB signaling pathway in endometrial cells. Our findings suggest that ADSC-Exo-based therapy might serve as an attractive strategy for the treatment of CE.

A study on bone marrow derived stem cells and its application on ACL graft regeneration in partial ACL tears

A study on bone marrow derived stem cells and its application on ACL graft regeneration in partial ACL tears

3D organic bioelectronics for electrical monitoring of human adult stem cells.

Three-dimensional in vitro stem cell models have enabled a fundamental understanding of cues that direct stem cell fate. While sophisticated 3D tissues can be generated, technology that can accurately monitor these complex models in a high-throughput and non-invasive manner is not well adapted. Here we show the development of 3D bioelectronic devices based on the electroactive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate)-(PEDOT:PSS) and their use for non-invasive, electrical monitoring of stem cell growth. We show that the electrical, mechanical and wetting properties as well as the pore size/architecture of 3D PEDOT:PSS scaffolds can be fine-tuned simply by changing the processing crosslinker additive. We present a comprehensive characterization of both 2D PEDOT:PSS thin films of controlled thicknesses, and 3D porous PEDOT:PSS structures made by the freeze-drying technique. By slicing the bulky scaffolds we generate homogeneous, porous 250 μm thick PEDOT:PSS slices, constituting biocompatible 3D constructs able to support stem cell cultures. These multifunctional slices are attached on indium-tin oxide substrates (ITO) with the help of an electrically active adhesion layer, enabling 3D bioelectronic devices with a characteristic and reproducible, frequency dependent impedance response. This response changes drastically when human adipose derived stem cells (hADSCs) grow within the porous PEDOT:PSS network as revealed by fluorescence microscopy. The increase of cell population within the PEDOT:PSS porous network impedes the charge flow at the interface between PEDOT:PSS and ITO, enabling the interface resistance (R1) to be used as a figure of merit to monitor the proliferation of stem cells. The non-invasive monitoring of stem cell growth allows for the subsequent differentiation 3D stem cell cultures into neuron like cells, as verified by immunofluorescence and RT-qPCR measurements. The strategy of controlling important properties of 3D PEDOT:PSS structures simply by altering processing parameters can be applied for development of a number of stem cell in vitro models as well as stem cell differentiation pathways. We believe the results presented here will advance 3D bioelectronic technology for both fundamental understanding of in vitro stem cell cultures as well as the development of personalized therapies.

Surfaceome mapping of primary human heart cells with CellSurfer uncovers cardiomyocyte surface protein LSMEM2 and proteome dynamics in failing hearts

Cardiac cell surface proteins are drug targets and useful biomarkers for discriminating among cellular phenotypes and disease states. Here we developed an analytical platform, CellSurfer, that enables quantitative cell surface proteome (surfaceome) profiling of cells present in limited quantities, and we apply it to isolated primary human heart cells. We report experimental evidence of surface localization and extracellular domains for 1,144 N-glycoproteins, including cell-type-restricted and region-restricted glycoproteins. We identified a surface protein specific for healthy cardiomyocytes, LSMEM2, and validated an anti-LSMEM2 monoclonal antibody for flow cytometry and imaging. Surfaceome comparisons among pluripotent stem cell derivatives and their primary counterparts highlighted important differences with direct implications for drug screening and disease modeling. Finally, 20% of cell surface proteins, including LSMEM2, were differentially abundant between failing and non-failing cardiomyocytes. These results represent a rich resource to advance development of cell type and organ-specific targets for drug delivery, disease modeling, immunophenotyping and in vivo imaging