Showing papers in "Stem Cells in 2015"
TL;DR: It is found that hucMSC‐Ex‐treated wounds exhibited significantly accelerated re‐epithelialization, with increased expression of CK19, PCNA, collagen I (compared to collagen III) in vivo.
Abstract: Mesenchymal stem cell-derived exosomes (MSC-Ex) play important roles in tissue injury repair, however, the roles of MSC-Ex in skin damage repair and its mechanisms are largely unknown. Herein, we examined the benefit of human umbilical cord MSC-derived exosome (hucMSC-Ex) in cutaneous wound healing using a rat skin burn model. We found that hucMSC-Ex-treated wounds exhibited significantly accelerated re-epithelialization, with increased expression of CK19, PCNA, collagen I (compared to collagen III) in vivo. HucMSC-Ex promoted proliferation and inhibited apoptosis of skin cells after heat-stress in vitro. We also discovered that Wnt4 was contained in hucMSC-Ex, and hucMSC-Ex-derived Wnt4 promoted β-catenin nuclear translocation and activity to enhance proliferation and migration of skin cells, which could be reversed by β-catenin inhibitor ICG001. In vivo studies confirmed that the activation of Wnt/β-catenin by hucMSC-Ex played a key role in wound re-epithelialization and cell proliferation. Furthermore, knockdown of Wnt4 in hucMSC-Ex abrogated β-catenin activation and skin cell proliferation and migration in vitro. The in vivo therapeutic effects were also inhibited when the expression of Wnt4 in hucMSC-Ex was interfered. In addition, the activation of AKT pathway by hucMSC-Ex was associated with the reduction of heat stress-induced apoptosis in rat skin burn model. Collectively, our findings indicate that exosome-delivered Wnt4 provides new aspects for the therapeutic strategy of MSCs in cutaneous wound healing. Stem Cells 2015;33:2158-2168.
563 citations
TL;DR: This review outlines the properties of various dental MSC‐like populations and the progress toward their use in regenerative therapy.
Abstract: Recently, numerous types of human dental tissue-derived mesenchymal stem cells (MSCs) have been isolated and characterized, including dental pulp stem cells, stem cells from exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle progenitor cells, alveolar bone-derived MSCs, stem cells from apical papilla, tooth germ progenitor cells, and gingival MSCs. All these MSC-like cells exhibit self-renewal, multilineage differentiation potential, and immunomodulatory properties. Several studies have demonstrated the potential advantages of dental stem cell-based approaches for regenerative treatments and immunotherapies. This review outlines the properties of various dental MSC-like populations and the progress toward their use in regenerative therapy. Several dental stem cell banks worldwide are also introduced, with a view toward future clinical application.
257 citations
TL;DR: The results demonstrated that the novel pathway H19/miR‐675/TGF‐β1/Smad3/HDAC regulates osteogenic differentiation of hMSCs and may serve as a potential target for enhancing bone formation in vivo.
Abstract: Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules at the transcriptional and post-transcriptional levels and may play essential roles in the differentiation of human bone marrow mesenchymal stem cell (hMSC). However, their roles and functions remain unclear. Here, we showed that lncRNA H19 was significantly upregulated after the induction of osteoblast differentiation. Overexpression of H19 promoted osteogenic differentiation of hMSCs in vitro and enhanced heterotopic bone formation in vivo, whereas knockdown of H19 inhibited these effects. Subsequently, we found that miR-675, encoded by exon1 of H19, promoted osteoblast differentiation of hMSCs and was partially responsible for the pro-osteogenic effect of H19. Investigating the underlying mechanism, we demonstrated that H19/miR-675 inhibited mRNA and protein expression of transforming growth factor-β1 (TGF-β1). The downregulation of TGF-β1 subsequently inhibited phosphorylation of Smad3. Meanwhile, H19/miR-675 downregulated the mRNA and protein levels of histone deacetylase (HDAC) 4/5, and thus increased osteoblast marker gene expression. Taken together, our results demonstrated that the novel pathway H19/miR-675/TGF-β1/Smad3/HDAC regulates osteogenic differentiation of hMSCs and may serve as a potential target for enhancing bone formation in vivo.
253 citations
TL;DR: It is shown that MSCs from MM expressed less lncRNA MEG3 relative to those from normal donors during osteogenic differentiation, suggesting that MEG 3 played an essential role in osteogenic differentiate in bone marrow MSC's, partly by activating BMP4 transcription.
Abstract: Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of mesenchymal stromal cells (MSCs). However, the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules in tumor-suppressor and oncogenic pathways. Here we showed that MSCs from MM expressed less lncRNA MEG3 relative to those from normal donors during osteogenic differentiation. To evaluate the effect of MEG3 on osteogenesis, bone marrow MSCs with enhanced or reduced MEG3 were prepared. We observed that MEG3 knockdown significantly reduced the expression of key osteogenic markers, including Runt-related transcription factor 2, osterix, and osteocalcin, while overexpression of MEG3 enhanced their expression. Additionally, MEG3 knockdown decreased BMP4 transcription. Here we showed that MEG3 was critical for SOX2 transcriptional repression of the BMP4. MEG3, which is located near the BMP4 gene, could dissociate the transcription factor SOX2 from the BMP4 promoter. A stable complex containing the MEG3, SOX2, and the SOX2 consensus site of BMP4 suggested that MEG3 activated transcriptional activity by directly influencing SOX2 activity. By using assays such as luciferase, chromatin immunoprecipitation, and RNA immunoprecipitation, we showed that MEG3 had a critical function in a mechanism of promoter-specific transcriptional activation. These results suggested that MEG3 played an essential role in osteogenic differentiation in bone marrow MSCs, partly by activating BMP4 transcription. Our data provided novel evidence for the biological and clinical significance of lncRNA MEG3 expression as a potential biomarker for identifying patients with MM and as a potential therapeutic target in MM.
224 citations
TL;DR: It is shown that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer, and this increased lipid content was clearly revealed by label‐free Raman spectroscopy and it directly correlates with well‐accepted CR‐CSC markers as CD133 and Wnt pathway activity.
Abstract: The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs. Stem Cells 2015;33:35–44
182 citations
TL;DR: The novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries is supported, as they can acquire multipotential stem cell activity and differentiate into major components of the BBB/neurovascular unit.
Abstract: Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.
182 citations
TL;DR: The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of M SCs prior to their therapeutic application.
Abstract: This study sought to identify critical determinants of mesenchymal stem cell (MSC) potency using in vitro and in vivo attributes of cells isolated from the bone marrow of age- and sex-matched donors. Adherence to plastic was not indicative of potency, yet capacity for long-term expansion in vitro varied considerably between donors, allowing the grouping of MSCs from the donors into either those with high-growth capacity or low-growth capacity. Using this grouping strategy, high-growth capacity MSCs were smaller in size, had greater colony-forming efficiency, and had longer telomeres. Cell-surface biomarker analysis revealed that the International Society for Cellular Therapy (ISCT) criteria did not distinguish between high-growth capacity and low-growth capacity MSCs, whereas STRO-1 and platelet-derived growth factor receptor alpha were preferentially expressed on high-growth capacity MSCs. These cells also had the highest mean expression of the mRNA transcripts TWIST-1 and DERMO-1. Irrespective of these differences, both groups of donor MSCs produced similar levels of key growth factors and cytokines involved in tissue regeneration and were capable of multilineage differentiation. However, high-growth capacity MSCs produced approximately double the volume of mineralized tissue compared to low-growth capacity MSCs when assessed for ectopic bone-forming ability. The additional phenotypic criteria presented in this study when combined with the existing ISCT minimum criteria and working proposal will permit an improved assessment of MSC potency and provide a basis for establishing the quality of MSCs prior to their therapeutic application.
174 citations
TL;DR: It is demonstrated that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than zinc finger nucleases and TALENs.
Abstract: Human induced pluripotent stem cells (iPSCs) and genome editing provide a precise way to generate gene-corrected cells for disease modeling and cell therapies. Human iPSCs generated from sickle cell disease (SCD) patients have a homozygous missense point mutation in the HBB gene encoding adult β-globin proteins, and are used as a model system to improve strategies of human gene therapy. We demonstrate that the CRISPR/Cas9 system designer nuclease is much more efficient in stimulating gene targeting of the endogenous HBB locus near the SCD point mutation in human iPSCs than ZFNs and TALENs. Using a specific guide RNA and Cas9, we readily corrected one allele of the SCD HBB gene in human iPSCs by homologous recombination with a donor DNA template containing the wild-type HBB DNA and a selection cassette that was subsequently removed to avoid possible interference of HBB transcription and translation. We chose targeted iPSC clones that have one corrected and one disrupted SCD allele for erythroid differentiation assays, using an improved xeno-free and feeder-free culture condition we recently established. Erythrocytes from either the corrected or its parental (uncorrected) iPSC line were generated with similar efficiencies. Currently ~6%-10% of these differentiated erythrocytes indeed lacked nuclei, characteristic of further matured erythrocytes called reticulocytes. We also detected the 16-kD β-globin protein expressed from the corrected HBB allele in the erythrocytes differentiated from genome-edited iPSCs. Our results represent a significant step towards the clinical applications of genome editing using patient-derived iPSCs to generate disease-free cells for cell and gene therapies.
172 citations
TL;DR: It is suggested that preincubation of MSCs for 2 days or more in hypoxia induces metabolic changes that yield higher retention after transplantation, and HP‐MSCs showed enhanced survival in vivo after intramuscular injection into immune deficient NOD/SCID‐IL2Rgamma−/− mice.
Abstract: Mesenchymal stem cells/multipotent stromal cells (MSCs) are promising therapeutics for a variety of conditions. However, after transplantation, cell retention remains extremely challenging. Given that many hypoxic signals are transitory and that the therapeutic administration of MSCs is typically into tissues that are normally hypoxic, we studied the effect of hypoxic preconditioning (HP) prior to new exposure to hypoxia. We show that preincubation for 2 days or more in 1% oxygen reduces serum deprivation-mediated cell death, as observed by higher cell numbers and lower incorporation of EthD-III and Annexin V. Consistently, HP-MSCs expressed significantly lower levels of cytochrome c and heme oxygenase 1 as compared to controls. Most importantly, HP-MSCs showed enhanced survival in vivo after intramuscular injection into immune deficient NOD/SCID-IL2Rgamma(-/-) mice. Interestingly, HP-MSCs consume glucose and secrete lactate at a slower rate than controls, possibly promoting cell survival, as glucose remains available to the cells for longer periods of time. In addition, we compared the metabolome of HP-MSCs to controls, before and after hypoxia and serum deprivation, and identified several possible mediators for HP-mediated cell survival. Overall, our findings suggest that preincubation of MSCs for 2 days or more in hypoxia induces metabolic changes that yield higher retention after transplantation.
171 citations
TL;DR: GA‐hMSCs increase proliferation and self‐renewal of GSCs in vitro and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche.
Abstract: Although mesenchymal stem cells (MSCs) have been implicated as stromal components of several cancers, their ultimate contribution to tumorigenesis and their potential to drive cancer stem cells, particularly in the unique microenvironment of human brain tumors, remain largely undefined. Consequently, using established criteria, we isolated glioma-associated-human MSCs (GA-hMSCs) from fresh human glioma surgical specimens for the first time. We show that these GA-hMSCs are nontumorigenic stromal cells that are phenotypically similar to prototypical bone marrow-MSCs. Low-passage genomic sequencing analyses comparing GA-hMSCs with matched tumor-initiating glioma stem cells (GSCs) suggest that most GA-hMSCs (60%) are normal cells recruited to the tumor (group 1 GA-hMSCs), although, rarely (10%), GA-hMSCs may differentiate directly from GSCs (group 2 GA-hMSCs) or display genetic patterns intermediate between these groups (group 3 GA-hMSCs). Importantly, GA-hMSCs increase proliferation and self-renewal of GSCs in vitro and enhance GSC tumorigenicity and mesenchymal features in vivo, confirming their functional significance within the GSC niche. These effects are mediated by GA-hMSC-secreted interleukin-6, which activates STAT3 in GSCs. Our results establish GA-hMSCs as a potentially new stromal component of gliomas that drives the aggressiveness of GSCs, and point to GA-hMSCs as a novel therapeutic target within gliomas.
167 citations
TL;DR: The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers as mentioned in this paper, which may underlie clinical manifestations of malignant disease.
Abstract: The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self-renewal and long-term proliferative potential of stem-like cancer cells, NANOG-mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development.
TL;DR: It is demonstrated that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B‐cell response in immune disease.
Abstract: Mesenchymal or stromal stem cells (MSC) interact with cells of the immune system in multiple ways. Modulation of the immune system by MSC is believed to be a therapeutic option for autoimmune disease and transplant rejection. In recent years, B cells have moved into the focus of the attention as targets for the treatment of immune disorders. Current B-cell targeting treatment is based on the indiscriminate depletion of B cells. The aim of this study was to examine whether human adipose tissue-derived MSC (ASC) interact with B cells to affect their proliferation, differentiation, and immune function. ASC supported the survival of quiescent B cells predominantly via contact-dependent mechanisms. Coculture of B cells with activated T helper cells led to proliferation and differentiation of B cells into CD19(+) CD27(high) CD38(high) antibody-producing plasmablasts. ASC inhibited the proliferation of B cells and this effect was dependent on the presence of T cells. In contrast, ASC directly targeted B-cell differentiation, independently of T cells. In the presence of ASC, plasmablast formation was reduced and IL-10-producing CD19(+) CD24(high) CD38(high) B cells, known as regulatory B cells, were induced. These results demonstrate that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B-cell response in immune disease.
TL;DR: This study provides evidence of safety and feasibility in the nonsurgical treatment of DDD with autologous BMC and indicates an effect of mesenchymal cell concentration on discogenic pain reduction.
Abstract: Degenerative disc disease (DDD) induces chronic back pain with limited nonsurgical options. In this open label pilot study, 26 patients (median age 40 years; range 18-61) received autologous bone marrow concentrate (BMC) disc injections (13 one level, 13 two levels). Pretreatment Oswestry disability index (ODI) and visual analog scale (VAS) were performed to establish baseline pain scores (average 56.5 and 79.3, respectively), while magnetic resonance imaging was independently scored according to the modified Pfirrmann scale. Approximately 1 ml of BMC was analyzed for total nucleated cell (TNC) content, colony-forming unit-fibroblast (CFU-F) frequency, differentiation potential, and phenotype characterization. The average ODI and VAS scores were reduced to 22.8 and 29.2 at 3 months, 24.4 and 26.3 at 6 months, and 25.0 and 33.2 at 12 months, respectively (p ≤ .0001). Eight of twenty patients improved by one modified Pfirrmann grade at 1 year. The average BMC contained 121 × 10(6) TNC/ml with 2,713 CFU-F/ml (synonymous with mesenchymal stem cells). Although all subjects presented a substantial reduction in pain, patients receiving greater than 2,000 CFU-F/ml experienced a significantly faster and greater reduction in ODI and VAS. Subjects older than 40 years who received fewer than 2,000 CFU-F/ml experienced an average pain reduction of 33.7% (ODI) and 29.1% (VAS) at 12 months, while all other patients' average reduction was 69.5% (ODI, p = .03) and 70.6% (VAS, p = .01). This study provides evidence of safety and feasibility in the nonsurgical treatment of DDD with autologous BMC and indicates an effect of mesenchymal cell concentration on discogenic pain reduction.
TL;DR: These studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures and report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors.
Abstract: The derivation of three-dimensional (3D) stratified neural retina from pluripotent stem cells has permitted investigations of human photoreceptors. We have generated a H9 human embryonic stem cell subclone that carries a green fluorescent protein (GFP) reporter under the control of the promoter of cone-rod homeobox (CRX), an established marker of postmitotic photoreceptor precursors. The CRXp-GFP reporter replicates endogenous CRX expression in vitro when the H9 subclone is induced to form self-organizing 3D retina-like tissue. At day 37, CRX+ photoreceptors appear in the basal or middle part of neural retina and migrate to apical side by day 67. Temporal and spatial patterns of retinal cell type markers recapitulate the predicted sequence of development. Cone gene expression is concomitant with CRX, whereas rod differentiation factor neural retina leucine zipper protein (NRL) is first observed at day 67. At day 90, robust expression of NRL and its target nuclear receptor NR2E3 is evident in many CRX+ cells, while minimal S-opsin and no rhodopsin or L/M-opsin is present. The transcriptome profile, by RNA-seq, of developing human photoreceptors is remarkably concordant with mRNA and immunohistochemistry data available for human fetal retina although many targets of CRX, including phototransduction genes, exhibit a significant delay in expression. We report on temporal changes in gene signatures, including expression of cell surface markers and transcription factors; these expression changes should assist in isolation of photoreceptors at distinct stages of differentiation and in delineating coexpression networks. Our studies establish the first global expression database of developing human photoreceptors, providing a reference map for functional studies in retinal cultures.
TL;DR: A novel regulation of stem‐like functions by YAP1, through the modulation of Sox2 expression is demonstrated, which prevents the growth and metastasis of tumor xenografts in mice.
Abstract: Non-small cell lung cancer (NSCLC) is highly correlated with smoking and has very low survival rates. Multiple studies have shown that stem-like cells contribute to the genesis and progression of NSCLC. Our results show that the transcriptional coactivator yes-associated protein 1 (YAP1), which is the oncogenic component of the Hippo signaling pathway, is elevated in the stem-like cells from NSCLC and contributes to their self-renewal and ability to form angiogenic tubules. Inhibition of YAP1 by a small molecule or depletion of YAP1 by siRNAs suppressed self-renewal and vascular mimicry of stem-like cells. These effects of YAP1 were mediated through the embryonic stem cell transcription factor, Sox2. YAP1 could transcriptionally induce Sox2 through a physical interaction with Oct4; Sox2 induction occurred independent of TEAD2 transcription factor, which is the predominant mediator of YAP1 functions. The binding of Oct4 to YAP1 could be detected in cell lines as well as tumor tissues; the interaction was elevated in NSCLC samples compared to normal tissue as seen by proximity ligation assays. YAP1 bound to Oct4 through the WW domain, and a peptide corresponding to this region could disrupt the interaction. Delivery of the WW domain peptide to stem-like cells disrupted the interaction and abrogated Sox2 expression, self-renewal, and vascular mimicry. Depleting YAP1 reduced the expression of multiple epithelial-mesenchymal transition genes and prevented the growth and metastasis of tumor xenografts in mice; overexpression of Sox2 in YAP1 null cells rescued these functions. These results demonstrate a novel regulation of stem-like functions by YAP1, through the modulation of Sox2 expression.
TL;DR: The subcutaneous application of NOD2‐activated hUCB‐MSCs can efficiently ameliorate AD, and MSC‐derived PGE2 and TGF‐β1 are required for the inhibition of MC degranulation.
Abstract: Mesenchymal stem cell (MSC) is a promising tool for the therapy of immune disorders. However, their efficacy and mechanisms in treating allergic skin disorders are less verified. We sought to investigate the therapeutic efficacy of human umbilical cord blood-derived MSCs (hUCB-MSCs) against murine atopic dermatitis (AD) and to explore distinct mechanisms that regulate their efficacy. AD was induced in mice by the topical application of Dermatophagoides farinae. Naive or activated-hUCB-MSCs were administered to mice, and clinical severity was determined. The subcutaneous administration of nucleotide-binding oligomerization domain 2 (NOD2)-activated hUCB-MSCs exhibited prominent protective effects against AD, and suppressed the infiltration and degranulation of mast cells (MCs). A β-hexosaminidase assay was performed to evaluate the effect of hUCB-MSCs on MC degranulation. NOD2-activated MSCs reduced the MC degranulation via NOD2-cyclooxygenase-2 signaling. In contrast to bone marrow-derived MSCs, hUCB-MSCs exerted a cell-to-cell contact-independent suppressive effect on MC degranulation through the higher production of prostaglandin E2 (PGE2 ). Additionally, transforming growth factor (TGF)-β1 production from hUCB-MSCs in response to interleukin-4 contributed to the attenuation of MC degranulation by downregulating FceRI expression in MCs. In conclusion, the subcutaneous application of NOD2-activated hUCB-MSCs can efficiently ameliorate AD, and MSC-derived PGE2 and TGF-β1 are required for the inhibition of MC degranulation.
TL;DR: Findings indicate that LGR5 not only participates in carcinogenesis but also maintained stemness by activating Wnt/β‐catenin signaling in breast cancer.
Abstract: The cancer stem cell (CSC) hypothesis suggests that a subset of cancer cells possesses stem cell properties and is crucial in tumor initiation, metastasis, and drug resistance. To determine the mechanism of CSCs in breast cancer, we focused on LGR5, a marker of adult stem cells that potentially serves as a functional factor in CSCs. LGR5 overexpression was detected in breast cancer and significantly associated with breast cancer recurrence and poor outcome. LGR5 promoted cell mobility, tumor formation, and epithelial-mesenchymal transition in breast cancer cells by activating Wnt/β-catenin signaling. In addition, LGR5 was more highly expressed in tumorspheres and increased the stemness of breast cancer cells. Compared with LGR5 low-expression (LGR5low) cells, LGR5high cells exhibited CSC/tumor-initiating cell-like properties, including the formation of self-renewing spheres and high tumorigenicity. Importantly, our studies indicate that LGR5 activation of Wnt/β-catenin signaling is a possible mechanism to regulate breast CSC/tumor-initiating cell renewal. These findings indicate that LGR5 not only participates in carcinogenesis but also maintained stemness by activating Wnt/β-catenin signaling in breast cancer. Stem Cells 2015;33:2913–2924
TL;DR: In mesenchymal stem cells (MSCs), ectopic Mkx expression strikingly promoted tenogenesis more efficiently than Scleraxis (Scx), a well‐known master transcription factor of tendon.
Abstract: The transcription factor Mohawk (Mkx) is expressed in developing tendons and is an important regulator of tenogenic differentiation. However, the exact roles of Mkx in tendinopathy and tendon repair remain unclear. Using gene expression Omnibus datasets and immunofluorescence assays, we found that Mkx expression level was dramatically lower in human tendinopathy tissue and it is activated at specific stages of tendon development. In mesenchymal stem cells (MSCs), ectopic Mkx expression strikingly promoted tenogenesis more efficiently than Scleraxis (Scx), a well-known master transcription factor of tendon. Significantly higher levels of tenogenic gene expression and collagen fibril growth were observed with Mkx versus Scx. Interestingly, it was observed that Mkx dramatically upregulated Scx through binding to the Tgfb2 promoter. Additionally, the transplantation of Mkx-expressing-MSC sheets promoted tendon repair in a mouse model of Achilles-tendon defect. Taken together, these data shed light on previously unrecognized roles of Mkx in tendinopathy, tenogenesis, and tendon repair as well as in regulating the TGFβ pathway.
TL;DR: This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound‐healing, disease, and following transplantation.
Abstract: Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreER(T2)-Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K-14(+) progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound-healing, disease, and following transplantation.
TL;DR: In this paper, the authors investigated whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts.
Abstract: Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) β, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells.
TL;DR: This study illustrates notable success in using the self‐assembling method to generate robust, scaffold‐free neocartilage constructs using expanded hACs and hMSCs.
Abstract: Replacement of degenerated cartilage with cell-based cartilage products may offer a long-term solution to halt arthritis' degenerative progression. Chondrocytes are frequently used in cell-based FDA-approved cartilage products; yet human marrow-derived stromal cells (hMSCs) show significant translational potential, reducing donor site morbidity and maintaining their undifferentiated phenotype with expansion. This study sought to investigate the effects of transforming growth factor β1 (TGF-β1), growth/differentiation factor 5 (GDF-5), and bone morphogenetic protein 2 (BMP-2) during postexpansion chondrogenesis in human articular chondrocytes (hACs) and to compare chondrogenesis in passaged hACs with that of passaged hMSCs. Through serial expansion, chondrocytes dedifferentiated, decreasing expression of chondrogenic genes while increasing expression of fibroblastic genes. However, following expansion, 10 ng/mL TGF-β1, 100 ng/mL GDF-5, or 100 ng/mL BMP-2 supplementation during three-dimensional aggregate culture each upregulated one or more markers of chondrogenic gene expression in both hACs and hMSCs. Additionally, in both cell types, the combination of TGF-β1, GDF-5, and BMP-2 induced the greatest upregulation of chondrogenic genes, that is, Col2A1, Col2A1/Col1A1 ratio, SOX9, and ACAN, and synthesis of cartilage-specific matrix, that is, glycosaminoglycans (GAGs) and ratio of collagen II/I. Finally, TGF-β1, GDF-5, and BMP-2 stimulation yielded mechanically robust cartilage rich in collagen II and GAGs in both cell types, following 4 weeks maturation. This study illustrates notable success in using the self-assembling method to generate robust, scaffold-free neocartilage constructs using expanded hACs and hMSCs.
TL;DR: In this paper, the authors provide an update regarding the biological mechanisms of adult stem cells quiescence and their role in tissue regeneration, and provide an overview of the role of stem cells in regeneration.
Abstract: Adult stem cells (ASCs) are tissue resident stem cells responsible for tissue homeostasis and regeneration following injury. In uninjured tissues, ASCs exist in a nonproliferating, reversibly cell cycle-arrested state known as quiescence or G0. A key function of the quiescent state is to preserve stemness in ASCs by preventing precocious differentiation, and thus maintaining a pool of undifferentiated ASCs. Recent evidences suggest that quiescence is an actively maintained state and that excessive or defective quiescence may lead to compromised tissue regeneration or tumorigenesis. The aim of this review is to provide an update regarding the biological mechanisms of ASC quiescence and their role in tissue regeneration.
TL;DR: It is shown that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK‐dependent induction of RhoA, indicating that the MSC responds to its dynamic physical environment not only with “outside‐in’ signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent “inside–inside” signaling.
Abstract: A cell’s ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSC), high magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of FAK/mTORC2/Akt signaling generated at focal adhesions [1]. Physiologic systems also rely on a persistent barrage of low level signals to regulate behavior [2]. Exposing MSC to extremely low magnitude mechanical signals (LMS) suppresses adipocyte formation [3] despite the virtual absence of substrate strain (<0.001%) [2], suggesting that LMS-induced dynamic accelerations can generate force within the cell. Here we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating focal adhesion kinase (FAK) and Akt signaling followed by FAK-dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the focal adhesions, LMS-induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear-actin cytoskeleton mechanocoupling by disrupting LINC (Linker of Nucleoskeleton and Cytoskeleton) complexes inhibited these LMS-induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by high magnitude strain (HMS) was unaffected by LINC decoupling, consistent with signal initiation at the focal adhesion (FA) mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with “outside-in” signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent “inside-inside” signaling.
TL;DR: It is reported that insulin‐like growth factor 1 (IGF‐1) can orchestrate the formation of three‐dimensional ocular‐like structures from hESCs which, in addition to retinal pigmented epithelium and neural retina, also contain primitive lens and corneal‐ like structures.
Abstract: We and others have previously demonstrated that retinal cells can be derived from human embryonic stem cells (hESCs) and induced pluripotent stem cells under defined culture conditions. While both cell types can give rise to retinal derivatives in the absence of inductive cues, this requires extended culture periods and gives lower overall yield. Further understanding of this innate differentiation ability, the identification of key factors that drive the differentiation process, and the development of clinically compatible culture conditions to reproducibly generate functional neural retina is an important goal for clinical cell based therapies. We now report that insulin-like growth factor 1 (IGF-1) can orchestrate the formation of three-dimensional ocular-like structures from hESCs which, in addition to retinal pigmented epithelium and neural retina, also contain primitive lens and corneal-like structures. Inhibition of IGF-1 receptor signaling significantly reduces the formation of optic vesicle and optic cups, while exogenous IGF-1 treatment enhances the formation of correctly laminated retinal tissue composed of multiple retinal phenotypes that is reminiscent of the developing vertebrate retina. Most importantly, hESC-derived photoreceptors exhibit advanced maturation features such as the presence of primitive rod- and cone-like photoreceptor inner and outer segments and phototransduction-related functional responses as early as 6.5 weeks of differentiation, making these derivatives promising candidates for cell replacement studies and in vitro disease modeling.
TL;DR: Targeting the tumor microenvironment, such as interrupting the immune cell, for example, myeloid‐derived suppressor cells, and cytokines, as well as the immune checkpoint (PD1/PDL1, etc.) may provide additional novel strategies to enhance the immunological targeting of CSCs.
Abstract: Cancer stem cells (CSCs) represent a small subset of tumor cells which have the ability to self-renew and generate the diverse cells that comprise the tumor bulk. They are responsible for local tumor recurrence and distant metastasis. However, they are resistant to conventional radiotherapy and chemotherapy. Novel immunotherapeutic strategies that specifically target CSCs may improve the efficacy of cancer therapy. To immunologically target CSC phenotypes, innate immune responses to CSCs have been reported using Natural killer cells and γδ T cells. To target CSC specifically, in vitro CSC-primed T cells have been successfully generated and shown targeting of CSCs in vivo after adoptive transfer. Recently, CSC-based dendritic cell vaccine has demonstrated significant induction of anti-CSC immunity both in vivo in immunocompetent hosts and in vitro as evident by CSC reactivity of CSC vaccine-primed antibodies and T cells. In addition, identification of specific antigens or genetic alterations in CSCs may provide more specific targets for immunotherapy. ALDH, CD44, CD133, and HER2 have served as markers to isolate CSCs from a number of tumor types in animal models and human tumors. They might serve as useful targets for CSC immunotherapy. Finally, since CSCs are regulated by interactions with the CSC niche, these interactions may serve as additional targets for CSC immunotherapy. Targeting the tumor microenvironment, such as interrupting the immune cell, for example, myeloid-derived suppressor cells, and cytokines, for example, IL-6 and IL-8, as well as the immune checkpoint (PD1/PDL1, etc.) may provide additional novel strategies to enhance the immunological targeting of CSCs.
TL;DR: 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation, and mechanical loading promotes cardiomyocyte structural and functional maturation.
Abstract: Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle, and endothelial cells. Because the heart develops from progenitors in 3D under constant mechanical load, we sought to test the effects of a 3D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2D culture, the unstressed 3D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3D culture suppressed smooth muscle α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including β-myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue. Stem Cells 2015;33:2148-2157.
TL;DR: It is proposed that in the authors' medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency, and this was demonstrated to be a fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.
Abstract: Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naive pluripotent state Efforts have been made to trigger naive pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines This required either ectopic expression of naive genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naive pluripotency in hESCs, as well as in mESCs, which has not been shown earlier The converted naive hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naive pluripotency genes, and exhibited dependence on signaling pathways similar to naive mESCs Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naive pluripotency Collectively, we demonstrate an alternate route to capture naive pluripotency in hESCs that is fast, reproducible, supports naive mESC derivation, and allows efficient differentiation
TL;DR: It is suggested that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal‐resident microglia and the activity and recruitment of hematogenous macrophages.
Abstract: Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C-γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (p-ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor-α, interleukin-6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte-macrophage colony-stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages.
TL;DR: Allo‐ASC therapy was safe and effective in improving elbow pain, performance, and structural defects for 52 weeks and is the first to reveal therapeutic value of mesenchymal stem cell injection for treating chronic tendinopathy.
Abstract: Mesenchymal stem cell therapy is a novel regenerative approach for treating tendinopathy. Here, we evaluated the safety and efficacy of allogeneic adipose-derived mesenchymal stem cells (allo-ASC) in treating lateral epicondylosis (LE). Under ultrasound guidance, allo-ASCs mixed with fibrin glue were injected into the hypoechoic common extensor tendon lesions of 12 participants with chronic LE; 6 subjects each were administered 106 or 107 cells in 1 ml. Safety was evaluated at day 3 and weeks 2, 6, 12, 26, and 52 post-injection. Efficacy was assessed by measuring patients’ visual analog scale (VAS) score for elbow pain, modified Mayo clinic performance index for the elbow, and by evaluating longitudinal and transverse ultrasound images of tendon defect areas after 6, 12, 26, and 52 weeks. No significant adverse effects of allo-ASC injection were observed through 52 weeks of follow-up. From baseline through 52 weeks of periodic follow-up, VAS scores progressively decreased from 66.8 ± 14.5 mm to 14.8 ± 13.1 mm and elbow performance scores improved from 64.0 ± 13.5 to 90.6 ± 5.8. Tendon defects also significantly decreased through this period. Allo-ASC therapy was thus safe and effective in improving elbow pain, performance, and structural defects for 52 weeks. This clinical study is the first to reveal therapeutic value of mesenchymal stem cell injection for treating chronic tendinopathy. Stem Cells 2015;33:2995–3005
TL;DR: It is demonstrated how expression of the biomarker panel is conserved in the human retina and proposed that a similar selection strategy will facilitate isolation of human transplantation‐competent cells for therapeutic application.
Abstract: Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.