Showing papers in "Stem Cells and Development in 2014"
TL;DR: It is demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active and induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue reporter cell line.
Abstract: Mesenchymal stem cells (MSCs) have been shown to secrete exosomes that are cardioprotective. Here, we demonstrated that MSC exosome, a secreted membrane vesicle, is immunologically active. MSC exosomes induced polymyxin-resistant, MYD88-dependent secreted embryonic alkaline phosphatase (SEAP) expression in a THP1-Xblue, a THP-1 reporter cell line with an NFκB-SEAP reporter gene. In contrast to lipopolysaccharide, they induced high levels of anti-inflammatory IL10 and TGFβ1 transcript at 3 and 72 h, and much attenuated levels of pro-inflammatory IL1B, IL6, TNFA and IL12P40 transcript at 3-h. The 3-h but not 72-h induction of cytokine transcript was abrogated by MyD88 deficiency. Primary human and mouse monocytes exhibited a similar exosome-induced cytokine transcript profile. Exosome-treated THP-1 but not MyD88-deficient THP-1 cells polarized activated CD4+ T cells to CD4+CD25+FoxP3+ regulatory T cells (Tregs) at a ratio of one exosome-treated THP-1 cell to 1,000 CD4+ T cells. Infusion of MSC exosomes enha...
515 citations
TL;DR: The NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.
Abstract: Human embryonic stem cells and induced pluripotent stem cells have great potential in research and therapies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here, we demonstrate, for the first time, that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4, NANOG, and SSEA-4, in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme, cellulase, enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly, the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus, the NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.
204 citations
TL;DR: Human adipose-derived stem cells produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for nerve regeneration, and their effects in a peripheral nerve injury model were determined.
Abstract: In future, adipose-derived stem cells (ASC) might be used to treat neurological disorders. In this study, the neurotrophic and angiogenic properties of human ASC were evaluated, and their effects i ...
178 citations
TL;DR: This is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2), and shows that hesenchymal stem cells have therapeutic efficacy in two different autoimmune disorder models.
Abstract: Mesenchymal stem cells (MSCs) are being tested in a wide range of human diseases; however, loss of potency and inconsistent quality severely limit their use. To overcome these issues, we have utilized a developmental precursor called the hemangioblast as an intermediate cell type in the derivation of a highly potent and replenishable population of MSCs from human embryonic stem cells (hESCs). This method circumvents the need for labor-intensive hand-picking, scraping, and sorting that other hESC-MSC derivation methods require. Moreover, unlike previous reports on hESC-MSCs, we have systematically evaluated their immunomodulatory properties and in vivo potency. As expected, they dynamically secrete a range of bioactive factors, display enzymatic activity, and suppress T-cell proliferation that is induced by either allogeneic cells or mitogenic stimuli. However, they also display unique immunophenotypic properties, as well as a smaller size and >30,000-fold proliferative capacity than bone marrow-derived MSCs. In addition, this is the first report which demonstrates that hESC-MSCs can inhibit CD83 up-regulation and IL-12p70 secretion from dendritic cells and enhance regulatory T-cell populations induced by interleukin 2 (IL-2). This is also the first report which shows that hESC-MSCs have therapeutic efficacy in two different autoimmune disorder models, including a marked increase in survival of lupus-prone mice and a reduction of symptoms in an autoimmune model of uveitis. Our data suggest that this novel and therapeutically active population of MSCs could overcome many of the obstacles that plague the use of MSCs in regenerative medicine and serve as a scalable alternative to current MSC sources.
146 citations
TL;DR: Human induced pluripotent stem cell lines from fibroblasts obtained from individuals with FXS are created to enable in vitro modeling of the human disease and provide a well-characterized resource to examine potential neuronal deficits caused by FXS as well as the function of FMRP in human neurons.
Abstract: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and is closely linked with autism. The genetic basis of FXS is an expansion of CGG repeats in the 5′-untranslated region of the FMR1 gene on the X chromosome leading to the loss of expression of the fragile X mental retardation protein (FMRP). The cause of FXS has been known for over 20 years, yet the full molecular and cellular consequences of this mutation remain unclear. Although mouse and fly models have provided significant understanding of this disorder and its effects on the central nervous system, insight from human studies is limited. We have created human induced pluripotent stem cell (iPSC) lines from fibroblasts obtained from individuals with FXS to enable in vitro modeling of the human disease. Three young boys with FXS who came from a well-characterized cohort representative of the range of affectedness typical for the syndrome were recruited to aid in linking cellular and behavioral phenotypes. The FMR1 mutation is preserved during the reprogramming of patient fibroblasts to iPSCs. Mosaicism of the CGG repeat length in one of the patient's fibroblasts allowed for the generation of isogenic lines with differing CGG repeat lengths from the same patient. FXS forebrain neurons were differentiated from these iPSCs and display defective neurite initiation and extension. These cells provide a well-characterized resource to examine potential neuronal deficits caused by FXS as well as the function of FMRP in human neurons.
140 citations
TL;DR: It is suggested that hematopoietic differentiation efficiency of β-Thal iPSC-derived HSCs in transplantation was greatly improved once corrected by the CRISPR/Cas9 system.
Abstract: The generation of beta-thalassemia (β-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the β-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected β-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct β-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected β-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected β-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of β-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of β-Thal iPSC-derived HSCs in transplantation.
135 citations
TL;DR: Control induction of iPSC into fibroblastic-like cells that phenotypically and functionally resemble adult MSC is an attractive approach to obtain a readily available source of progenitor cells for orthopedic and dental-related tissue-engineering applications.
Abstract: The therapeutic potential of mesenchymal stem cells (MSC) has highlighted the need for identifying easily accessible and reliable sources of these cells. An alternative source for obtaining large populations of MSC is through the controlled differentiation of induced pluripotent stem cells (iPSC). In the present study, colonies of iPSC were cultured in MSC culture media for 2 weeks. Serial passaging then selected for fast growing MSC-like cells with a typical fibroblastic morphology and the capacity to proliferate on standard culture flasks without feeder cells. MSC-like cells were developed from iPSC lines arising from three different somatic tissues: gingiva, periodontal ligament (PDL), and lung. The iPSC-MSC like cells expressed key MSC-associated markers (CD73, CD90, CD105, CD146, and CD166) and lacked expression of pluripotent markers (TRA160, TRA181, and alkaline phosphatase) and hematopoietic markers (CD14, CD34, and CD45). In vitro iPSC-MSC-like cells displayed the capacity to differentiate into osteoblasts, adipocytes, and chondrocytes. In vivo subcutaneous implantation of the iPSC-MSC-like cells into NOD/SCID mice demonstrated that only the PDL-derived iPSC-MSC-like cells exhibited the capacity to form mature mineralized structures which were histologically similar to mature bone. These findings demonstrate that controlled induction of iPSC into fibroblastic-like cells that phenotypically and functionally resemble adult MSC is an attractive approach to obtain a readily available source of progenitor cells for orthopedic and dental-related tissue-engineering applications. However, a detailed characterization of the iPSC-MSC-like cells will be important, as MSC-like cells derived from different iPSC lines exhibit variability in their differentiation capacity.
134 citations
TL;DR: A novel role of let-7/HMGA2 axis is found in regulating the balance of osteogenesis and adipogenesis of MSCs and can be used as a novel therapeutic target for disorders that are associated with bone loss and adipocyte accumulation.
Abstract: Bone and fat cells share a common progenitor, stromal/mesenchymal stem cells (MSCs), that can differentiate into osteoblasts or adipocytes. Osteogenesis and adipogenesis of MSCs maintain homeostasi...
130 citations
TL;DR: iMPCs generated from PSCs via spontaneous differentiation represent a distinct population of cells which exhibit MSC-like characteristics, and are less responsive to traditional BMSC differentiation protocols.
Abstract: Mesenchymal stem cells (MSCs) have a high potential for therapeutic efficacy in treating diverse musculoskeletal injuries and cardiovascular diseases, and for ameliorating the severity of graft-versus-host and autoimmune diseases. While most of these clinical applications require substantial cell quantities, the number of MSCs that can be obtained initially from a single donor is limited. Reports on the derivation of MSC-like cells from pluripotent stem cells (PSCs) are, thus, of interest, as the infinite proliferative capacity of PSCs opens the possibility to generate large amounts of uniform batches of MSCs. However, characterization of such MSC-like cells is currently inadequate, especially with regard to the question of whether these cells are equivalent or identical to MSCs. In this study, we have derived MSC-like cells [induced PSC-derived MSC-like progenitor cells (iMPCs)] using four different methodologies from a newly established induced PSC line reprogrammed from human bone marrow stromal cells ...
127 citations
TL;DR: Adipose-Muse cells distinguish themselves as both easily obtainable and versatile in their capacity for differentiation, while low telomerase activity and lack of teratoma formation make these cells a practical cell source for potential stem cell therapies, particularly for ectodermal and endodermal tissues where transplanted cells need to differentiate across the lineage.
Abstract: In this study, we demonstrate that a small population of pluripotent stem cells, termed adipose multilineage-differentiating stress-enduring (adipose-Muse) cells, exist in adult human adipose tissue and adipose-derived mesenchymal stem cells (adipose-MSCs). They can be identified as cells positive for both MSC markers (CD105 and CD90) and human pluripotent stem cell marker SSEA-3. They intrinsically retain lineage plasticity and the ability to self-renew. They spontaneously generate cells representative of all three germ layers from a single cell and successfully differentiate into targeted cells by cytokine induction. Cells other than adipose-Muse cells exist in adipose-MSCs, however, do not exhibit these properties and are unable to cross the boundaries from mesodermal to ectodermal or endodermal lineages even under cytokine inductions. Importantly, adipose-Muse cells demonstrate low telomerase activity and transplants do not promote teratogenesis in vivo. When compared with bone marrow (BM)– a...
124 citations
TL;DR: The results revealed that the HuMenSCs could survive within POF mouse ovaries for at least 14 days in vivo, and the mesenchymal stem cell properties and in vivo survival of Hu menSCs make them ideal seed cells for stem cell transplantation in the treatment of POF.
Abstract: The incidence of premature ovarian failure (POF), also known as ovarian insufficiency, has been increasing in recent years. Although some treatments are currently available, improved treatment strategies are urgently required. Many researchers have reported that human endometrial stem cells (HuMenSCs), which exhibit stem/progenitor cell properties in vitro repaired damaged cells in vivo. Thus, we aimed to determine whether HuMenSCs can serve as cell therapy tools and be used for the treatment of POF. After treating with cyclophosphamide, on the first estrus period (we predicted mouse estrus cycle was generally 5 days), HuMenSCs were injected into a cyclophosphamide-induced mouse model of POF. The results revealed that the HuMenSCs could survive within POF mouse ovaries for at least 14 days in vivo; further, ovaries of the HuMenSCs-transplanted group expressed higher levels of ovarian markers [AMH, inhibin α/β, and follicle-stimulating hormone receptor (FSHR)], and the proliferative marker Ki67. In addition, the ovarian weight, plasma E2 level, and the number of normal follicles increased over time in the HuMenSC group compared with the control group. Further, microarray analysis of cDNA expression patterns revealed that, after HuMenSC transplantation, the gene mRNA expression patterns in the ovarian cells following stimulation of the host ovarian niche became increasingly similar to those observed in human ovarian tissue compared with the pretransplantation mRNA expression pattern in HuMenSCs. Hence, we can safely conclude that the mesenchymal stem cell properties and in vivo survival of HuMenSCs make them ideal seed cells for stem cell transplantation in the treatment of POF.
TL;DR: This is the first report to demonstrate the self-renewal capacity of PDLSCs using serial xenogeneic transplants and provides evidence of the long-term survival and tissue contribution of autologous PDL SCs in a preclinical periodontal defect model.
Abstract: Primary periodontal ligament stem cells (PDLSCs) are known to possess multidifferentiation potential and exhibit an immunophenotype similar to that described for bone-marrow-derived mesenchymal stem cells. In the present study, bromo-deoxyuridine (BrdU)–labeled ovine PDLSCs implanted into immunodeficient mice survived after 8 weeks post-transplantation and exhibited the capacity to form bone/cementum-like mineralized tissue, ligament structures similar to Sharpey's fibers with an associated vasculature. To evaluate self-renewal potential, PDLSCs were recovered from harvested primary transplants 8 weeks post-transplantation that exhibit an immunophenotype and multipotential capacity comparable to primary PDLSCs. The re-derived PDLSCs isolated from primary transplants were implanted into secondary ectopic xenogeneic transplants. Histomorphological analysis demonstrated that four out of six donor re-derived PDLSC populations displayed a capacity to survive and form fibrous ligament structures and mineralized...
TL;DR: Investigation of the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion indicates that M SCs may transfer and modulate the expression of several mi RNAs involved in the repair and recovery process in PTECs.
Abstract: The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.
TL;DR: The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a growing number of companies established.
Abstract: The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a growing number of companies established. The field, nevertheless, faces several challenges. Persistent issues include the definition of a MSC and comparability between MSC preparations. This is because of inherent cell heterogeneity, the absence of markers that are unique to MSCs, and the difficulty in precisely defining them by developmental origin. Differences in the properties of MSCs also depend on the site of tissue harvest, phenotypic and genotypic characteristics of the donor and the isolation, and storage and expansion methods used. These differences may be sufficient to ensure that attributes of the final MSC product could differ in potentially significant ways. Since there are currently no gold standards, we propose using a reference material to establish methods of comparability among MSC preparations. We suggest four possible "ruler scenarios" and a method for global distribution. We further suggest that critical to establishing a reference material is the need to define protocols for comparing cells. The main purpose of this article is to solicit input in establishing a consensus-based comparison. A comparative approach will be critical to all stages of translation to better clarify mechanisms of MSC actions, define an optimal cell manufacturing process, ensure best practice clinical investigations, extend the use of an MSC product for new indications, protect an MSC product from imitators, and develop uniform reimbursement policies. Importantly, a reference material may enable a consensus on a practical definition of MSCs.
TL;DR: Data indicate a lower in vitro immunomodulatory effect of MVs on T-cell proliferation and antibody formation, as compared with their cellular counterpart, which needs to be compared in proper prospective studies.
Abstract: Mesenchymal stromal cells (MSCs) are multipotent cells that possess broad immunomodulatory properties; the mechanisms underlying these properties have not been completely clarified. Aim of this study was to compare in vitro immunomodulatory effects of MSCs with those of microvesicles (MVs) released in supernatants from the same MSCs. MSCs were generated from bone marrow of 12 healthy donors (HDs) and MVs were isolated from their supernatant by serial ultracentrifugation according to two different procedures. Both MSCs and MVs were characterized by flow cytometry and incubated in vitro with peripheral blood mononuclear cells (PBMCs) of 12 HDs after stimulation with PHA and CpG. Growth factors and cytokines were quantified by ELISA. MVs were identified as 0.1-1 μm particles positive for CMFDA, CD107, and CD13. MSCs were significantly more capable to inhibit in vitro PHA-induced T-cell proliferation as compared with the corresponding MVs (P<0.01 and P<0.05 for MSC:PBMC ratio 1:2 and 1:10, respectively). While MVs displayed similar inhibitory activity on B-cell proliferation (P=0.43 as compared with PBMCs/CpG/MSCs; MSC:PBMC ratio 1:10) they induced lower inhibitory effect on plasmacell differentiation and antibody secretion (P<0.05 as compared with PBMCs/CpG/MSCs). For both T and B cells, MSC co-colture induced a statistically significant increase in IL-10 and TGFβ and decrease of GM-CSF and IFNγ, as compared with MV incubation. Our data indicate a lower in vitro immunomodulatory effect of MVs on T-cell proliferation and antibody formation, as compared with their cellular counterpart. The relative clinical benefit of either MSCs or MVs needs to be compared in proper prospective studies.
TL;DR: Interestingly, SOX9 and aggrecan proteins were found to be downregulated in anti-miR-140 transduced differentiating MSCs despite unchanged mRNA levels, which suggests that miR- 140 stimulates in vitro chondrogenesis by the upregulation of these molecules at the protein level.
Abstract: Lesions of articular cartilage do not heal spontaneously. One treatment strategy would be to make cartilage in the laboratory by directed chondrogenic differentiation of mesenchymal stem cells (MSCs). To promote our understanding of the molecular control of chondrogenesis, we have compared the changes in microRNAs (miRNAs) during in vitro chondrogenesis of MSCs with those observed in uncultured and dedifferentiated articular chondrocytes (ACs). Several miRNAs showed a reciprocal relationship during the differentiation of MSCs and dedifferentiation of ACs. miR-140-5p and miR-140-3p changed the most during in vitro chondrogenesis, they were the miRNAs most highly expressed in tissue-engineered chondrocytes, and they were also among the miRNAs most highly expressed in uncultured ACs. There was a 57% overlap for the 100 most highly expressed miRNAs in differentiated MSCs and uncultured ACs, but for other miRNAs, the expression pattern was quite different. We transiently and stably inhibited and overexpressed miR-140-5p and miR-140-3p in differentiating MSCs and dedifferentiating ACs, respectively, to describe global effects and identify and validate new targets. Surprisingly, SOX9 and aggrecan proteins were found to be downregulated in anti-miR-140 transduced differentiating MSCs despite unchanged mRNA levels. This suggests that miR-140 stimulates in vitro chondrogenesis by the upregulation of these molecules at the protein level. RALA, a small GTPase, was identified as a miR-140 target and knockdown experiments showed that RALA regulated SOX9 at the protein level. These observations shed new light on the effect of miR-140 for chondrogenesis in vitro and in vivo.
TL;DR: The results suggest that MSCs can functionally deliver exogenous miRNAs to neural cells and provide an efficient route of therapeutic miRNA delivery to the brain in pathological conditions with clinical implications for regenerative medicine.
Abstract: MicroRNAs (miRNAs) are potential therapeutic targets in a variety of pathological conditions in the brain; however, their clinical application is hampered by lack of efficient delivery modes. Mesenchymal stromal stem cells (MSCs) migrate to sites of injury and inflammation and exert therapeutic effects in various neurological disorders. Here, we examined the ability of MSCs to deliver exogenous miRNA mimics and pre-miRNAs to human neural progenitor cells (NPCs) and astrocytes and characterized the functional impact of this delivery. We found that MSCs efficiently delivered fluorescent-labeled miR-124 and miR-145 mimics to cocultured NPCs and astrocytes. We further demonstrated the delivery of the miRNAs using novel reporter plasmids that contain a sequence complementary to miR-124 or miR-145 downstream of luciferase or mCherry. Binding of the specific miRNAs to these sequences results in decreased luciferase activity or mCherry fluorescence and therefore enable analysis of miRNA delivery in living cells. The delivered exogenous miR-124 significantly decreased the expression of the target gene Sox9 by targeting its 3'-UTR, and increased the neuronal differentiation of the NPCs. In addition, the delivered miR-124 increased the expression of the glutamate transporters, EAAT1 in NPCs and EAAT2 in both NPCs and astrocytes. Similar results were obtained with MSCs transfected with pre-miR-124. The miRNA delivery was mediated by MSC-derived exosomes and was cell contact independent. These results suggest that MSCs can functionally deliver exogenous miRNAs to neural cells and provide an efficient route of therapeutic miRNA delivery to the brain in pathological conditions with clinical implications for regenerative medicine.
TL;DR: A highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell and human induced PSC lines tested, and the present yield and efficiency was up to 5-fold higher and without the need of performing line-specific optimization.
Abstract: Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to β-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.
TL;DR: It is shown that an inflammatory environment triggered by lipopolysaccharide in vitro would suppress BMP-2-induced osteogenic differentiation of bone marrow mesenchymal stem cells, and that the crosstalk between TLR4/MyD88/NF-κB and BMP/Smad signaling negatively modulates the osteoinductive capacity of B MP-2.
Abstract: Bone morphogenetic protein-2 (BMP-2) is a novel differentiation factor that is capable of inducing osteoblast differentiation and bone formation, making it an attractive option in treatment of bone defects, fractures, and spine fusions. Inflammation, which was a common situation during bone healing, is recognized to inhibit osteogenic differentiation and bone formation. However, the effect of inflammation on BMP-2-induced osteoblastic differentiation remains ambiguous. In this study, we showed that an inflammatory environment triggered by lipopolysaccharide (LPS) in vitro would suppress BMP-2-induced osteogenic differentiation of bone marrow mesenchymal stem cells, which represented by decreased alkaline phosphatase (ALPase) activity and down-regulated osteogenic genes. In addition, LPS activated nuclear factor-κB (NF-κB) via a TLR4/MyD88-dependent manner and inhibited BMP-2-induced phosphorylation and nuclear translocation of Smad1/5/8. The blocking of NF-κB signaling by pretreatment with specific inhibitors such as BAY-11-7082, TPCK and PDTC, or by transfection with plasmids encoding p65 siRNA or IκBα siRNA could significantly reverse the inhibitory effect of LPS on BMP-2-induced BMP/Smad signaling and osteogenic differentiation. By contrast, even without stimulation of LPS, overexpression of p65 gene showed obvious inhibitory effects on BMP-2-induced BMP/Smad signaling and ALPase activity. These data indicate that the LPS-mediated inflammatory environment inhibits BMP-2-induced osteogenic differentiation, and that the crosstalk between TLR4/MyD88/NF-κB and BMP/Smad signaling negatively modulates the osteoinductive capacity of BMP-2.
TL;DR: The concept of totipotency is discussed, and the confusions surrounding this term in the scientific and nonscientific literature are considered, and a new term, "plenipotent," is proposed to resolve this confusion.
Abstract: There is surprising confusion surrounding the concept of biological totipotency, both within the scientific community and in society at large. Increasingly, ethical objections to scientific research have both practical and political implications. Ethical controversy surrounding an area of research can have a chilling effect on investors and industry, which in turn slows the development of novel medical therapies. In this context, clarifying precisely what is meant by “totipotency” and how it is experimentally determined will both avoid unnecessary controversy and potentially reduce inappropriate barriers to research. Here, the concept of totipotency is discussed, and the confusions surrounding this term in the scientific and nonscientific literature are considered. A new term, “plenipotent,” is proposed to resolve this confusion. The requirement for specific, oocyte-derived cytoplasm as a component of totipotency is outlined. Finally, the implications of twinning for our understanding of totipotency are d...
TL;DR: It is concluded that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.
Abstract: In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.
TL;DR: This review seeks to shed further light on interactions between MSCs and endothelial cells in different physiopathological conditions and reports both capacity and differentiation into endothelial-like cells have been reported in MSC-based pro-angiogenic therapies.
Abstract: Recent years have witnessed the emergence of a considerable amount of data pertaining to the application of bone marrow mesenchymal stem cells (MSCs) in promoting angiogenesis in the field of regenerative medicine. Nevertheless, some authors have provided evidence that MSCs can also prevent the process of angiogenesis, which is desirable in certain pathologies such as tumor growth. Plenty of in vitro and in vivo research studies have been undertaken to illuminate the underlying mechanisms by which MSCs promote or inhibit neo-angiogenesis. To date, both secretary capacity and differentiation into endothelial-like cells have been reported in MSC-based pro-angiogenic therapies. This review seeks to shed further light on interactions between MSCs and endothelial cells in different physiopathological conditions.
TL;DR: It was found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology, and should also become a useful biomaterial for studies on Parkinson's disease.
Abstract: The possibility to generate dopaminergic (DA) neurons from pluripotent stem cells represents an unlimited source of material for tissue engineering and cell therapy for neurodegenerative disease. We set up a protocol based on the generation of size-calibrated neurospheres for a rapid production (3 weeks) of a high amount of DA neurons (>60%) oriented toward a midbrain-like phenotype, characterized by the expression of FOXA2, LMX1A, tyrosine hydroxylase (TH), NURR1, and EN1. By using γ-secretase inhibitors and varying culture time of neurospheres, we controlled maturation and cellular composition of a three-dimensional (3D) engineered nervous tissue (ENT). ENT contained neurons and glial cells expressing various markers of maturity, such as synaptophysin, neuronal nuclei-specific protein (NeuN), and glial fibrillary acidic protein (GFAP), and were electrophysiologically active. We found that 3-week-old neurospheres were optimal to generate 3D tissue containing DA neurons with typical A9 morphology. ENT generated from 4-week-old neurospheres launched glial cell type since astrocytes and myelin could be detected massively at the expense of TH-immunoreactive neurons. All γ-secretase inhibitors were not equivalent; compound E was more efficient than DAPT in generating DA neurons. This DA tissue provides a tool for drug screening, and toxicology. It should also become a useful biomaterial for studies on Parkinson's disease.
TL;DR: This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.
Abstract: Generation of induced pluripotent stem cells (iPSCs) via the ectopic expression of reprogramming factors is a simple, advanced, yet often perplexing technology due to low efficiency, slow kinetics, and the use of numerous distinct systems for factor delivery. Scientists have used almost all available approaches for the delivery of reprogramming factors. Even the well-established retroviral vectors confuse some scientists due to different tropisms in use. The canonical virus-based reprogramming poses many problems, including insertional mutagenesis, residual expression and re-activation of reprogramming factors, uncontrolled silencing of transgenes, apoptosis, cell senescence, and strong immunogenicity. To eliminate or alleviate these problems, scientists have tried various other approaches for factor delivery and transgene removal. These include transient transfection, nonintegrating viral vectors, Cre-loxP excision of transgenes, excisable transposon, protein transduction, RNA transfection, microRNA transfection, RNA virion, RNA replicon, nonintegrating replicating episomal plasmids, minicircles, polycistron, and preintegration of inducible reprogramming factors. These alternative approaches have their own limitations. Even iPSCs generated with RNA approaches should be screened for possible transgene insertions mediated by active endogenous retroviruses in the human genome. Even experienced researchers may encounter difficulty in selecting and using these different technologies. This survey presents overviews of iPSC technologies with the intention to provide a quick yet comprehensive reference for both new and experienced reprogrammers.
TL;DR: This work inhibited interleukin-6, nitric oxide, and prostaglandin E2 to determine their impact on stimulated T cell proliferation and determined how equine MSCs modulate lymphocyte proliferation either via cell cycle arrest or apoptosis.
Abstract: Mesenchymal stem cells (MSCs) are used in both human clinical trials and veterinary medicine for the treatment of inflammatory and immune-mediated diseases. MSCs modulate inflammation by decreasing the cells and products of the inflammatory response. Stimulated equine MSCs from bone marrow (BM), adipose tissue (AT), cord blood (CB), and umbilical cord tissue (CT) inhibit lymphocyte proliferation and decrease inflammatory cytokine production. We hypothesized that equine MSCs inhibit T cell proliferation through secreted mediators and that MSCs from different tissue sources decrease T cell proliferation through different mechanisms. To test our hypotheses, we inhibited interleukin-6 (IL-6), nitric oxide (NO), and prostaglandin E2 (PGE2) to determine their impact on stimulated T cell proliferation. We also determined how equine MSCs modulate lymphocyte proliferation either via cell cycle arrest or apoptosis. Inhibition of IL-6 or NO did not reverse the immunomodulatory effect of MSCs on activated T cells. In...
TL;DR: The derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase is reported, and these cells were derived from POMPs with lens epithelial cell–conditioned medium.
Abstract: The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell–conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated c...
TL;DR: The regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewal of esophageal cancer stem-like cells and promote the development of new therapeutic strategies and efficient drugs that target ESCC stem- like cells.
Abstract: Cancer stem-like cells exist in many malignancies and several stem cell-related genes and microRNAs, such as Bmi-1 and miR-203, have been identified as cancer stem-like cell regulators using gene microarray or sequencing analysis. Previously, we used side population (SP) sorting to enrich cancer stem-like cells from esophageal squamous cell carcinoma (ESCC) cell line EC9706. Our results demonstrated that EC9706 SP cells shared common features of cancer stem-like cells. In this study, we examined the expression of Bmi-1 and miR-203 in ESCC SP and non-SP (NSP) cells. Our results showed that, when compared with NSP cells, Bmi-1 was up-regulated and miR-203 was down-regulated in SP cells. During the differentiation from SP to NSP cells, the expression levels of Bmi-1 were gradually decreased. Overexpression of miR-203 resulted in a significant reduction of endogenous Bmi-1 protein level in EC9706 cells. SP and NSP analyses revealed that the SP cell fraction was markedly decreased in miR-203 overexpressed cells. miR-203 overexpressed cells also showed a significant reduction in colony formation, which was resistant to chemotherapeutic drug treatment and tumorigenicity in nude mice. Rescue experiments demonstrated that ectopic expression of Bmi-1 in miR-203 overexpressed cells increased the SP fraction and restored cell proliferation. Taken together, these results indicated that stem renewal factor Bmi-1 was a direct target of miR-203. The regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewal of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs that target ESCC stem-like cells.
TL;DR: Overall, evidence suggested that MSCs increased disc space height in the majority of animal models, and short-term MSC transplantation is safe and effective; however, additional, larger, and higher-quality studies are needed to assess the long-term safety and efficacy.
Abstract: Intervertebral disc degeneration is associated with low-back pain. Mesenchymal stem cells (MSCs) have been used to "regenerate" the disc. The aim of this study was to perform a systematic review of comparative controlled studies that have assessed the safety and efficacy of using MSCs for disc regeneration. Literature databases were extensively searched. Trial design, subject-type, MSC sources, injection method, disc assessment, outcome intervals, and complication events were assessed. Validity of each study was performed. Twenty-four animal studies were included with 20.8% of the studies reporting randomization of groups. Trials in humans fulfilling inclusion criteria were not noted. The studies represented 862 discs that were injected with MSCs and 1,603 discs as controls. All three types of MSCs (ie, bone marrow, synovial, and adipose tissues) showed successful inhibition of disc degeneration. Bone-marrow-derived MSCs demonstrated superior quality of repair compared with other non-MSC treatments. A 2.7% overall complication rate was noted, whereby complications were noted only in rabbits. Overall, evidence suggested that MSCs increased disc space height in the majority of animal models. This is the first systematic review to assess the safety and efficacy of MSCs for the treatment of disc degeneration. Short-term MSC transplantation is safe and effective; however, additional, larger, and higher-quality studies are needed to assess the long-term safety and efficacy. Inconsistencies in methodological design and outcome parameters prevent any robust conclusions. Human-based clinical trials are needed. Recommendations are further made to improve efficacy, reduce potential complications, and standardize techniques for future studies.
TL;DR: It is determined that Gal-9 expression could serve as a marker to predict a higher or lower immune modulatory potential of single cell preparations and therefore to distinguish the therapeutic potency of MSCs derived from different donors.
Abstract: Therapeutic approaches using multipotent mesenchymal stromal cells (MSCs) are advancing in regenerative medicine, transplantation, and autoimmune diseases. The mechanisms behind MSC immune modulation are still poorly understood and the prediction of the immune modulatory potential of single MSC preparations remains a major challenge for possible clinical applications. Here, we highlight galectin-9 (Gal-9) as a novel, important immune modulator expressed by MSCs, which is strongly upregulated upon activation of the cells by interferon-γ (IFN-γ). Further, we demonstrate that Gal-9 is a major mediator of the anti-proliferative and functional effects of MSCs not only on T cells but also on B cells. Here, Gal-9 and activated MSCs contribute to the suppression of antigen triggered immunoglobulin release. Moreover, we determined that Gal-9 expression could serve as a marker to predict a higher or lower immune modulatory potential of single cell preparations and therefore to distinguish the therapeutic potency of MSCs derived from different donors. Also in vivo co-administration of MSCs or murine Gal-9 resulted in significantly reduced IgG titers in mice immunized with human coagulation factor VIII (FVIII). In conclusion, Gal-9 acts as an immune modulator interfering with multiple cell types including B cells and Gal-9 may serve as a predictive indicator for clinical MSC therapy.
TL;DR: The results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation, which may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering.
Abstract: Dental pulp/dentin regeneration using dental stem cells combined with odontogenic factors may offer great promise to treat and/or prevent premature tooth loss. We previously demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most potent factors in inducing bone formation. Here, we investigate whether BMP9 can effectively induce odontogenic differentiation of the stem cells from mouse apical papilla (SCAPs). Using a reversible immortalization system expressing SV40 T flanked with Cre/loxP sites, we demonstrate that the SCAPs can be immortalized, resulting in immortalized SCAPs (iSCAPs) that express mesenchymal stem cell markers. BMP9 upregulates Runx2, Sox9, and PPARγ2 and odontoblastic markers, and induces alkaline phosphatase activity and matrix mineralization in the iSCAPs. Cre-mediated removal of SV40 T antigen decreases iSCAP proliferation. The in vivo stem cell implantation studies indicate that iSCAPs can differentiate into bone, cartilage, and, to lesser extent, adipocytes upon BMP9 stimulation. Our results demonstrate that the conditionally iSCAPs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages, including osteo/odontoblastic differentiation. Thus, the reversibly iSCAPs may serve as an important tool to study SCAP biology and SCAP translational use in tooth engineering. Further, BMP9 may be explored as a novel and efficacious factor for odontogenic regeneration.