Bone regeneration by systemic transplantation of mesenchymal stem cells (MSCs) is problematic due to the inability to control the MSCs’ commitment, growth and differentiation into functional osteoblasts on the bone surface. Our research group has developed a method to direct the MSCs to the bone surface by conjugating a synthetic peptidomimetic ligand (LLP2A) that has high affinity for activated α4β1 integrin on the MSC surface, with a bisphosphonates (alendronate) that has high affinity for bone (LLP2A-Ale), to direct the transplanted MSCs to bone. Our in vitro experiments demonstrated that mobilization of LLP2A-Ale to hydroxyapatite accelerated MSC migration that was associated with an increase in the phosphorylation of Akt kinase and osteoblastogenesis. LLP2A-Ale increased the homing of the transplanted MSCs to bone as well as the osteoblast surface, significantly increased the rate of bone formation and restored both trabecular and cortical bone loss induced by estrogen deficiency or advanced age in mice. These results support LLP2A-Ale as a novel therapeutic option to direct the transplanted MSCs to bone for the treatment of established bone loss related to hormone deficiency and aging.
Sox17 is essential for both endoderm development and fetal hematopoietic stem cell (HSC) maintenance. While endoderm-derived organs are well known to originate from Sox17-expressing cells it is less certain whether fetal HSCs also originate from Sox17-expressing cells. By generating a Sox17GFPCre allele and using it to assess the fate of Sox17-expressing cells during embryogenesis we confirmed that both endodermal and a part of definitive hematopoietic cells are derived from Sox17-positive cells. Prior to E9.5 the expression of Sox17 is restricted to the endoderm lineage. However, at E9.5 Sox17 is expressed in the endothelial cells (ECs) at the para-aortic splanchnopleural (P-Sp) region that contribute to the formation of HSCs at a later stage. The identification of two distinct progenitor cell populations that express Sox17 at E9.5 was confirmed using FACS together with RNA-Seq to determine the gene expression profiles of the two cell populations. Interestingly, this analysis revealed differences in the RNA processing of the Sox17 mRNA during embryogenesis. Taken together, these results indicate that Sox17 is expressed in progenitor cells derived from two different germ layers, further demonstrating the complex expression pattern of this gene and suggesting caution when using Sox17 as a lineage-specific marker.
Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth-permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells de-differentiate to a progenitor/stem cell phenotype to promote axonal re-growth. In this study, we show that Skin-derived precursors (SKPs), a recently identified neural-crest related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro 3-D cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis.
Cutaneous nerve regeneration; SKPs; Skin-derived precursors; Peripheral nerve regeneration; Neurofibromatosis Type 1; NF1; Neurofibroma; Neurofibromatosis; Schwann cells; Cutaneous nerve homeostasis
Although the concept of cancer stem cells (CSCs) is well accepted for many tumors, the existence of such cells in human melanoma has been the subject of debate. In the present study, we demonstrate the existence of human melanoma cells that fulfill the criteria for CSCs (self-renewal and differentiation) by serially xenotransplanting cells into NOD/SCID mice. These cells possess high aldehyde dehydrogenase (ALDH) activity with ALDH1A1 and ALDH1A3 being the predominant ALDH isozymes. ALDH-positive melanoma cells are more tumorigenic than ALDH-negative cells in both NOD/SCID mice and NSG mice. Biological analyses of the ALDH-positive melanoma cells reveal the ALDH isozymes to be key molecules regulating the function of these cells. Silencing ALDH1A by siRNA or shRNA leads to cell cycle arrest, apoptosis and decreased cell viability in vitro and reduced tumorigenesis in vivo. ALDH-positive melanoma cells are more resistant to chemotherapeutic agents and silencing ALDH1A by siRNA sensitizes melanoma cells to drug-induced cell death. Furthermore, we, for the first time, examined the molecular signatures of ALDH-positive CSCs from patient-derived tumor specimens. The signatures of melanoma CSCs include retinoic acid (RA)-driven target genes with RA response elements and genes associated with stem cell function. These findings implicate that ALDH isozymes are not only biomarkers of CSCs but also attractive therapeutic targets for human melanoma. Further investigation of these isozymes and genes will enhance our understanding of the molecular mechanisms governing CSCs and reveal new molecular targets for therapeutic intervention of cancer.
melanoma; cancer stem cells; tumor initiating cells; aldehyde dehydrogenase; microarray analysis; molecular targeted therapy
Culturing cells in 3D provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti-inflammatory properties. Here we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium from hMSC spheroids inhibited LPS-stimulated macrophages from secreting pro-inflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. Conditioned medium also increased the secretion of anti-inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti-inflammatory activity in conditioned medium had a small molecular weight, and microarray data suggested that it was PGE2. This was confirmed by the observations that PGE2 levels were markedly elevated in hMSC spheroid-conditioned medium, and that the anti-inflammatory activity was abolished by an inhibitor of COX-2, a silencing RNA for COX-2, and an antibody to PGE2. The anti-inflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti-inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFκB activation in the hMSCs. The results indicated that hMSCs in 3D-spheroid cultures are self-activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily pro-inflammatory M1 phenotype to a more anti-inflammatory M2 phenotype.
MSC; COX2; LPS; macrophage; spheroid; caspase
Transplantation of myogenic stem cells possesses great potential for long-term repair of dystrophic muscle. However, a single donor muscle biopsy is unlikely to provide enough cells to effectively transplant the muscle mass of a patient affected by muscular dystrophy. Expansion of cells ex vivo using traditional culture techniques significantly reduces engraftment potential. We hypothesized that activation of Notch signaling during ex vivo expansion would maintain donor cell engraftment potential. In this study, we expanded freshly isolated canine muscle-derived cells on tissue culture plates coated with Delta-1ext-IgG to activate Notch signaling or with human IgG as a control. A model of canine-to-murine xenotransplantation was used to quantitatively compare canine muscle cell engraftment, and determine if engrafted donor cells could function as satellite cells in vivo. We show that Delta-1ext-IgG inhibited differentiation of canine muscle-derived cells, and increased the level of genes normally expressed in myogenic precursors. Moreover, cells expanded on Delta-1ext-IgG resulted in a significant increase in the number of donor-derived fibers, as compared to cells expanded on human IgG, reaching engraftment levels similar to freshly isolated cells. Importantly, cells expanded on Delta-1ext-IgG engrafted to the recipient satellite cell niche, and contributed to further regeneration. A similar strategy of expanding human muscle-derived cells on Notch ligand might facilitate engraftment and muscle regeneration for patients affected with muscular dystrophy.
striated muscle; satellite cells; regeneration; primary cell culture; Notch ligand; cell transplantation
Transsynaptic circuit tracing using genetically modified Rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it has become possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem (ES) cells in vitro, and these properties are under intense investigation towards devising cell replacement therapies. Here, we report the generation of a novel mouse ES cell (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ES cell-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to functionally connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest.
monosynaptic; ES cells; retrograde; TVA; Rabies-G; circuit; synapse
The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker SSEA-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the down-regulation of two microRNAs (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of microRNA mimics and inhibitors of these two microRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by microRNAs in hESCs.
human embryonic stem cells; ATP-binding cassette; ABCG2; BMP-4; differentiation
T-box 3 (Tbx3) is a member of the T-box family of genes. Mutations that result in the haploinsufficiency of TBX3 cause Ulnar Mammary Syndrome (UMS) in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self-renewal and in their differentiation into extra-embryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs has not been explored. Since mouse and human ESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in human ESC self-renewal and differentiation. Using over-expression and knockdown strategies, we found that TBX3 over-expression promotes human ESC proliferation possibly by repressing the expression of both NFκBIB and p14ARF, known cell cycle regulators. During differentiation, TBX3 knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers (PAX6, LHX2, FOXG1, RAX). Taken together, our data suggests a role for TBX3 in human ESC proliferation and reveals an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self-renewal and differentiation in both human and mouse ESCs.
TBX3; human embryonic stem cells; self-renewal; differentiation; neuroepithelial
Pharmacological targeting of breast cancer stem cells (CSCs) is highly promising for the treatment of breast cancer, as the small population of CSCs appears responsible for tumor initiation and progression and also for resistance to conventional treatment. Here we report that the novel phosphosulindac (OXT-328, PS) selectively and effectively eliminates breast CSCs both in vitro and in vivo. PS reduced cell proliferaition and induced apoptosis in various breast CSCs. Breast CSCs are resistant to conventional cancer drugs but are sensitive to PS. Long-term treatment with PS of mixtures of cultured breast CSCs with breast cancer cells preferentially eliminated the CSCs. PS impaired the ability of CSCs to form mammospheres and markedly suppressed the expression of CSC-related genes. More importantly, PS prevented by half (p=0.06) the formation of tumors initiated by CSCs in immunodeficient mice, and inhibited by 83% (p<0.05) the growth of already formed breast cancer xenografts, reducing the proportion of CSCs in them. PS suppressed the Wnt/β-catenin pathway by stimulating the degradation of β-catenin and its relocalization to the cell membrane; and also blocked the epithelial-mesenchymal transition (EMT) and the generation of breast CSCs. These results indicate that PS has a strong inhibitory effect against breast cancer, acting, at least in part, by targeting CSCs through a signaling mechanism involving Wnt signaling.
CSCs; phosphosulindac; Wnt/β-catenin; EMT; breast cancer
Embryonic stem cell (ESC) identity and self-renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here, we show that three members of the Ccr4-Not complex, Cnot1, Cnot2, and Cnot3, play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts, these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs, Cnot1, Cnot2, and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that sup ports the ground state pluripotency. Genetic analysis indicated that they do not act through known self-renewal pathways or core transcription factors. Instead, they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly, these Cnot genes are also necessary for the maintenance of human ESCs, and silencing them mainly lead to TE and primitive endoderm differ entiation. Together, our results indicate that Cnot1, Cnot2, and Cnot3 represent a novel component of the core self-renewal and pluripotency circuitry conserved in mouse and human ESCs.
Ccr4-Not; Embryonic stem cell; Self-renewal; Pluripotency; Extraembryonic differentiation
Adult stem cell therapies have provided success for more than 50 years, through reconstitution of the hematopoietic system using bone marrow, umbilical cord blood, and mobilized peripheral blood transplantation. Mesenchymal stem cell (MSC)-mediated therapy is a fast-growing field that has proven safe and effective in the treatment of various degenerative diseases and tissue injuries. Since the first derivation of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), there has been impressive progress toward developing safe clinical applications from PSCs. Recent successes in transgene-free iPSC reprogramming have brought attention to the potential of clinical applications of these pluripotent cells, but key hurdles must be overcome, which are discussed in this review. Looking to the future, it could be advantageous to derive MSC from iPSC or human ESC in cases where genetic engineering is needed, since in the PSCs, clones with “safe harbor” vector integration could be selected, expanded, and differentiated. Here, we describe the status of the progress of the use of MSC and PSCs in clinical trials and analyze the challenges that should be overcome before iPSC-derived MSC therapy can be used widely in the clinic.
Mesenchymal stem cells; Induced pluripotent stem cells; Cellular therapy; Clinical trials; Regenerative medicine
A key mechanism for mesenchymal stem cells/bone marrow stromal cells (MSCs) to promote tissue repair is by secretion of soluble growth factors (GFs). Therefore, clinical application could be optimized by a combination of cell and gene therapies, where MSCs are genetically modified to express higher levels of a specific factor. However, it remains unknown how this overexpression may alter the fate of the MSCs. Here, we show effects of overexpressing the growth factors, such as basic fibroblast growth factor (bFGF), platelet derived growth factor B (PDGF-BB), transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor (VEGF), in human bone marrow-derived MSCs. Ectopic expression of bFGF or PDGF-B lead to highly proliferating MSCs and lead to a robust increase in osteogenesis. In contrast, adipogenesis was strongly inhibited in MSCs overexpressing PDGF-B and only mildly affected in MSCs overexpressing bFGF. Overexpression of TGF-β1 blocked both osteogenic and adipogenic differentiation while inducing the formation of stress fibers and increasing the expression of the smooth muscle marker calponin-1 and the chondrogenic marker collagen type II. In contrast, MSCs overexpressing VEGF did not vary from control MSCs in any parameters, likely due to the lack of VEGF receptor expression on MSCs. MSCs engineered to overexpress VEGF strongly induced the migration of endothelial cells and enhanced blood flow restoration in a xenograft model of hind limb ischemia. These data support the rationale for genetically modifying MSCs to enhance their therapeutically relevant trophic signals, when safety and efficacy can be demonstrated, and when it can be shown that there are no unwanted effects on their proliferation and differentiation.
Growth factors; Mesenchymal stem cells; Bone marrow stromal cells; Angiogenesis
Induced pluripotent stem cell technology has attracted enormous interests for potential application in regenerative medicine. Here, we reported that a specific glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021, can induce the reprogramming of mouse embryonic fibroblasts (MEFs) transduced by only Oct4 and Klf4 two factors. When combined with Parnate (also named tranylcypromine), an inhibitor of lysine-specific demethylase 1, CHIR99021 can result in the reprogramming of human primary keratinocyte transducted with Oct4 and Klf4 two factors. To our knowledge, this is the first time to generate human iPS cells from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK-3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming.
TP63 is required for preservation of epithelial regenerative stasis and regulates the activity of diverse genetic pathways; however, specific effector pathways are poorly understood. Data presented here indicate that reciprocal regulatory interactions between hedgehog signaling and TP63 mediate stage-specific effects on proliferation and clonigenicity of separable enriched mammary stem and progenitor fractions. Analysis of ΔN-p63 and TA-p63 indicates segregated expression in mammary stem and progenitor fractions, respectively, demonstrating that differential TP63 promoter selection occurs during elaboration of mammary progenitors by mammary stem cells. This segregation underlies mammary progenitor-specific expression of Indian Hedgehog, identifying it as a binary transcriptional target of TP63. Hedgehog activation in vivo enhances elaboration of mammary progenitors and decreases label retention within mammary stem cell-enriched fractions, suggesting that hedgehog exerts a mitogenic effect on mammary stem cells. Hedgehog signaling promotes differential TP63 promoter usage via disruption of Gli3 or Gli3R accumulation, and shRNA-mediated disruption of Gli3 expression was sufficient to alter TP63 promoter usage and enhance clonigenicity of mammary stem cells. Finally, hedgehog signaling is enhanced during pregnancy, where it contributes to expansion of the mammary progenitor compartment. These studies support a model in which hedgehog activates elaboration and differentiation of mammary progenitors via differential TP63 promoter selection and forfeiture of self-renewing capacity.
Mammary stem cells; Mammary progenitors; Quiescence; TP63; Hedgehog; Gli3
Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM-5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM-5-positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM-5-positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM-5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM-5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barrett’s esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. The GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin.
Endoderm; Cell surface antigen; Liver progenitor; Barrett’s esophagus; FCGBP; Pancreatic cancer
Synchronous with massive shifts in reproductive hormones, the uterus and its lining the endometrium expand to accommodate a growing fetus during pregnancy. In the absence of an embryo the endometrium, composed of epithelium and stroma, undergoes numerous hormonally regulated cycles of breakdown and regeneration. The hormonally mediated regenerative capacity of the endometrium suggests that signals that govern the growth of endometrial progenitors must be regulated by estrogen and progesterone. Here we report an antigenic profile for isolation of mouse endometrial epithelial progenitors. These cells are EpCAM+CD44+ITGA6hiThy1−PECAM1−PTPRC−Ter119−, comprise a minor subpopulation of total endometrial epithelia and possess a gene expression profile that is unique and different from other cells of the endometrium. The epithelial progenitors of the endometrium could regenerate in vivo, undergo multi-lineage differentiation and proliferate. We show that the number of endometrial epithelial progenitors is regulated by reproductive hormones. Co-administration of estrogen and progesterone dramatically expanded the endometrial epithelial progenitor cell pool. This effect was not observed when estrogen or progesterone was administered alone. Despite the remarkable sensitivity to hormonal signals, endometrial epithelial progenitors do not express estrogen or progesterone receptors. Therefore their hormonal regulation must be mediated through paracrine signals resulting from binding of steroid hormones to the progenitor cell niche. Discovery of signaling defects in endometrial epithelial progenitors or their niche can lead to development of better therapies in diseases of the endometrium.
The majority of human malignancies are believed to have epithelial origin, and the progression of cancer is often associated with a transient process named epithelial-mesenchymal transition (EMT). EMT is characterized by the loss of epithelial markers and the gain of mesenchymal markers that are typical of “cancer stem-like cells, ” which results in increased cell invasion and metastasis in vivo. Therefore, it is important to uncover the mechanistic role of factors that may induce EMT in cancer progression. Studies have shown that platelet-derived growth factor (PDGF) signaling contributes to EMT, and more recently, PDGF-D has been shown to regulate cancer cell invasion and angiogenesis. However, the mechanism by which PDGF-D promotes invasion and metastases and whether it is due to the acquisition of EMT phenotype remain elusive. For this study, we established stably transfected PC3 cells expressing high levels of PDGF-D, which resulted in the significant induction of EMT as shown by changes in cellular morphology concomitant with the loss of E-cadherin and zonula occludens-1 and gain of vimentin. We also found activation of mammalian target of rapamycin and nuclear factor-κB, as well as Bcl-2 overexpression, in PDGF-D PC3 cells, which was associated with enhanced adhesive and invasive behaviors. More importantly, PDGF-D-overexpressing PC3 cells showed tumor growth in SCID mice much more rapidly than PC3 cells. These results provided a novel mechanism by which PDGF-D promotes EMT, which in turn increases tumor growth, and these results further suggest that PDGF-D could be a novel therapeutic target for the prevention and/or treatment of prostate cancer.
Platelet-derived growth factor-D; Epithelial-mesenchymal transition; Mammalian target of rapamycin; Nuclear factor-κB; E-cadherin; Vimentin
During spinal cord development, progenitors in the neural tube are arranged within spatial domains that generate specific cell types. The ependyma of the post-natal spinal cord seems to retain cells with properties of the primitive neural stem cells, some of which are able to react to injury with active proliferation. However, the functional complexity and organization of this stem cell niche in mammals remains poorly understood. Here, we combined immunohistochemistry for cell-specific markers with patch-clamp recordings to test the hypothesis that the ependyma of the neonatal rat spinal cord contains progenitor-like cells functionally segregated within specific domains. Cells on the lateral aspects of the ependyma combined morphological and molecular traits of ependymocytes and radial glia (RG) expressing S100β and vimentin, displayed passive membrane properties and were electrically coupled via Cx43. Cells contacting the ventral and dorsal poles expressed the neural stem cell markers nestin and/or vimentin, had the typical morphology of RG and appeared uncoupled displaying various combinations of K+ and Ca2+ voltage-gated currents. Although progenitor-like cells were mitotically active around the entire ependyma, the proliferative capacity seemed higher on lateral domains. Our findings represent the first evidence that the ependyma of the rat harbors progenitor-like cells with heterogeneous electrophysiological phenotypes organized in spatial domains. The manipulation of specific functional properties in the heterogeneous population of progenitor-like cells contacting the ependyma may in a future help to regulate their behavior and lineage potential, providing the cell types required for the endogenous repair of the injured spinal cord.
progenitor cells; nestin; vimentin; patch-clamp; ependyma; spinal cord
Goblet cells are terminally differentiated cells secreting mucins and anti-bacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow-cycling corneal progenitor cells, proliferating cells, non-proliferating cells, and post-mitotic differentiated K12+Muc5ac+goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency.
tissue regeneration; tissue-specific stem cells; cell migration; stem cell microenvironment
The goal of this study was to determine the role of integrin-mediated adhesion of bone-marrow-derived progenitor cells (BMPCs) as a requirement for the endothelial barrier protection in a lung injury model. C57BL mice were used as the source for BMPCs, which were characterized as CD34+ and fetal liver kinase-1 (Flk1)+ and also an expression of a repertoire of integrins. We used a mouse model of bacterial lipopolysaccharide (LPS)-induced lung vascular injury and edema formation to test the effects of BMPC integrin expression in preventing endothelial barrier injury. Adhesion of BMPCs to purified extracellular matrix proteins induced focal adhesion kinase (Fak) phosphorylation and formation of branching point structures in a α4 and α5 integrin-dependent manner. BMPCs expressing red fluorescent protein (RFP) were administered via the retro-orbital venous route in mice treated intraperitonially with LPS (7.5 mg/kg body weight). We observed increased retention of RFP-labeled Flk1+ and CD34+ BMPCs for up to 8 weeks in mice injured with LPS. BMPC transplantation increased survival by 50% (at 72–96 hours after LPS) and reduced lung vascular injury and extravascular water content induced by LPS. However, blocking with anti-α4 or anti-α5 integrin antibody or shRNA-mediated silencing of α4 or α5 integrins in donor BMPCs failed to prevent the vascular injury or edema formation and mortality. Thus, α4 and α5 integrin-dependent adhesion of BMPCs in lung tissue plays a critical role in preventing lung vascular injury and increasing survival in a mouse model of LPS-induced acute lung injury.
Acute lung injury; Endothelial progenitor cells; Endothelial barrier; Integrins; Stem Cells
Human pluripotent stem cell-derived cardiomyocytes (hPS-CM) may offer a number of advantages over previous cardiac models, however, questions of their immaturity complicate their adoption as a new in vitro model. hPS-CM differ from adult cardiomyocytes with respect to structure, proliferation, metabolism and electrophysiology, better approximating fetal cardiomyocytes. Time in culture appears to significantly impact phenotype, leading to what can be referred to as early and late hPS-CM. This work surveys the phenotype of hPS-CM, including structure, bioenergetics, sensitivity to damage, gene expression, and electrophysiology, including action potential, ion channels, and intracellular calcium stores, while contrasting fetal and adult CM with hPS-CM at early and late time points after onset of differentiation.
Cardiomyocyte; Pluripotent stem cells; Phenotype; In vitro cell culture; Maturity
Expression of the four transcription factors, that is, Oct4, Sox2, cMyc, and Klf4 has been shown to generate induced pluripotent stem cells (iPSCs) from many types of specialized differentiated somatic cells. It remains unclear, however, whether fully committed skeletal muscle progenitor cells (myoblasts) have the potency to undergo reprogramming to develop iPSCs in line with previously reported cases. To test this, we have isolated genetically marked myoblasts derived from satellite cell of adult mouse muscles using the Cre-loxP system (Pax7-CreER:R26R and Myf5-Cre:R26R). On infection with retroviral vectors expressing the four factors, these myoblasts gave rise to myogenic lineage tracer lacZ-positive embryonic stem cell (ESC)-like colonies. These cells expressed ESC-specific genes and were competent to differentiate into all three germ layers and germ cells, indicating the successful generation of myoblast-derived iPSCs. Continuous expression of the MyoD gene, a master transcription factor for skeletal muscle specification, inhibited this reprogramming process in myoblasts. In contrast, reprogramming myoblasts isolated from mice lacking the MyoD gene led to an increase in reprogramming efficiency. Our data also indicated that Oct4 acts as a transcriptional suppressor of MyoD gene expression through its interaction with the upstream enhancer region. Taken together, these results indicate that suppression of MyoD gene expression by Oct4 is required for the initial reprogramming step in the development of iPSCs from myoblasts. This data suggests that the skeletal muscle system provides a well-defined differentiation model to further elaborate on the effects of iPSC reprogramming in somatic cells.
Induced pluripotent stem cell; Satellite cell; MyoD; Oct4; Skeletal muscle; Myoblast
The Notch pathway plays a pivotal role in regulating cell fate decisions in many stem cell systems. However, the full repertoire of Notch target genes in vivo and the mechanisms through which this pathway activity is integrated with other signaling pathways are largely unknown. Here, we report a transgenic mouse in which the activation of the Notch pathway massively expands the neural stem cell (NSC) pool in a cell context-dependent manner. Using this in vivo system, we identify direct targets of RBPJ/N1ICD in cortical NSCs at a genome-wide level through combined ChIP-Seq and transcriptome analyses. Through a highly conservative analysis of these data sets, we identified 98 genes that are directly regulated by N1ICD/RPBJ in vivo. These include many transcription factors that are known to be critical for NSC self-renewal (Sox2, Pax6, Tlx, and Id4) and the transcriptional effectors of the Wnt, SHH and Hippo pathways, TCF4, Gli2, Gli3, Yap1, and Tead2. Since little is known about the function of the Hippo-Yap pathway in NSCs, we analyzed Yap1 expression and function in NSCs. We show that Yap1 expression is restricted to the stem cell compartment in the developing forebrain and that its expression is sufficient to rescue Notch pathway inhibition in NSC self-renewal assays. Together, the results of this study reveal a previously underappreciated complexity and breadth of Notch1 targets in vivo and show direct interaction between Notch and Hippo-Yap pathways in NSCs.
Neural stem cells; Notch pathway; Hippo-Yap pathway; transcriptional targets; ChIP-seq
By sequentially applying sonic hedgehog (C25II) and CHIR99021 (GSK3β inhibitor) to induce the midbrain floor plate progenitors and fibroblast growth factor 8 (FGF8) to promote dopaminergic differentiation in a chemically defined medium, we have established a robust system for generation of midbrain dopamine (DA) neurons from human and rhesus monkey embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We found that CHIR99021 specifies diencephalon to hindbrain fates in a concentration-dependent manner and only a narrow concentration range of CHIR99021 at a particular window is necessary to induce the midbrain floor plate progenitors, expressing Corin, En1, FoxA2 and Lmx1a. FGF8 enhances the dopaminergic fate of the progenitors, thus generating DA neurons with midbrain characteristics, including expression of TH, Lmx1a/b, FoxA2, FoxP1, Nurr1 and En1 as well as typical electrophysiological properties. More than half of these DA neurons expressed A9 DA neuron markers Girk2 and ALDH1a1. The new strategy will allow generation of enriched populations of functional midbrain DA neurons from both human and monkey PSCs for disease modeling, drug testing, and potential cell therapy.
Parkinson’s disease; drug discovery; neural patterning; transplantation