Mesenchymal Progeny Research Articles

Connecting secretome to hematopoietic stem cell phenotype shifts in an engineered bone marrow niche.

Hematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct coculture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial least squares regression (PLSR), along with an iterative filter method, identified TGFβ-1, MMP-3, c-RP and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52±3.65-fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplifies hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route toward identifying feeder-free culture platforms for HSC expansion. Insight Hematopoietic stem cells within an artificial niche receive maintenance cues in the form of soluble factors from hematopoietic and mesenchymal progeny. Applying a proteomic regression analysis, we identify a reduced set of soluble factors correlated to maintenance of a hematopoietic phenotype during culture in a biomaterial model of the bone marrow niche. We identify a minimum factor cocktail that promotes hematopoietic maintenance potential in a gelatin-based culture, regardless of the presence of mesenchymal feeder cells. By combining empirical and computational methods, we report an experimentally feasible number of factors from a large dataset, enabling exogenous integration of soluble factors into an engineered hematopoietic stem cell for enhanced maintenance potential of a quiescent stem cell population.

DNA-PKcs modulates progenitor cell proliferation and fibroblast senescence in idiopathic pulmonary fibrosis

BackgroundRecent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types.MethodsSurgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4+ cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441.ResultsDNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4+ mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4+ mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4+ CgA+ expressing cells.ConclusionsTogether, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4+ mesenchymal progenitors, and the senescence of their mesenchymal progeny.

Role of Periostin in Cardiac Valve Development.

Although periostin plays a significant role in adult cardiac remodeling diseases, the focus of this review is on periostin as a valvulogenic gene. Periostin is expressed throughout valvular development, initially being expressed in endocardial endothelial cells that have been activated to transform into prevalvular mesenchyme termed "cushion tissues" that sustain expression of periostin throughout their morphogenesis into mature (compacted) valve leaflets. The phenotype of periostin null indicates that periostin is not required for endocardial transformation nor the proliferation of its mesenchymal progeny but rather promotes cellular behaviors that promote migration, survival (anti-apoptotic), differentiation into fibroblastic lineages, collagen secretion and postnatal remodeling/maturation. These morphogenetic activities are promoted or coordinated by periostin signaling through integrin receptors activating downstream kinases in cushion cells that activate hyaluronan synthetase II (Akt/PI3K), collagen synthesis (Erk/MapK) and changes in cytoskeletal organization (Pak1) which regulate postnatal remodeling of cells and associated collagenous matrix into a trilaminar (zonal) histoarchitecture. Pak1 binding to filamin A is proposed as one mechanism by which periostin supports remodeling. The failure to properly remodel cushions sets up a trajectory of degenerative (myxomatous-like) changes that over time reduce biomechanical properties and increase chances for prolapse, regurgitation or calcification of the leaflets. Included in the review are considerations of lineage diversity and the role of periostin as a determinant of mesenchymal cell fate.

BMP2 expression in the endocardial lineage is required for AV endocardial cushion maturation and remodeling

Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5–11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.

An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells.

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.

Abstract IA08: Developmental insights into breast cancer intratumoral heterogeneity

Abstract Breast cancer is no longer considered a single disease, but instead is made up of multiple subtypes with genetically and epigenetically heterogeneous tumors composed of numerous clones. Both the hierarchical cancer stem cell and clonal evolution models have been invoked to help explain this intratumoral heterogeneity. Several recent studies have helped define the functional interactions among the different cellular subpopulations necessary for the evolution of this complex ecosystem. These interactions involve paracrine interactions that include locally acting Wnt family members, reminiscent of the signaling pathways important for normal mammary gland development and stem cell self-renewal. For example, mammary gland stem cell self-renewal is regulated by paracrine signaling involving Wnt4 and RSPO1 secreted by differentiated mammary luminal cells under the influence of systemic steroid hormones. Furthermore, interactions between non-canonical and canonical Wnt pathways play an important role in mammary gland development and cell fate determination [1]. Using a genetically engineered p53 null triple negative breast cancer model, we described a novel feedback loop in which the breast cancer stem cells(CSC) function through the action of paracrine mediators secreted by the CSC progeny[2], similar to what is observed in the normal mammary gland. We demonstrated the existence of cross-talk between tumor cells via Wnt2, Cxcl12 and IL6 signaling, indicating the non-cell-autonomous properties and importance of cooperativity between tumor subpopulations in tumor initiation and progression. Ligands expressed in the mesenchymal progeny cells produced through the asymmetric division of CSCs interacted with receptors on the CSCs. Knockdown of the ligands in the mesenchymal cells and their respective receptors in the CSCs resulted in decreased mammosphere formation in co-cultures. Furthermore, knockdown of Wnt 2 in the mesenchymal cells resulted in increased tumor latency when limiting numbers of CSCs were transplanted into the cleared mammary fat pad. The functional interactions between CSCs and mesenchymal population of cells may provide an improved niche microenvironment, especially for those tumors initiated from extremely low numbers of the CSCs. These studies indicate that similar regulatory mechanisms may exist for the maintenance of both normal and tumor stem cells with respect to their symmetric and asymmetric division. A better understanding of these interactions, especially in the metastatic setting, will be important for the development of improved combinatorial therapies designed to prevent relapse and to ultimately decrease mortality.

Human Retinal Pigment Epithelium Stem Cell (RPESC).

The retinal pigment epithelium (RPE) is a pigmented cellular monolayer that supports photoreceptor cells located in the overlying neural retina. The RPE is critical for vision and its dysfunction results in numerous pathologies, several with limited available disease-altering strategies. Regeneration of the retina from RPE is robust in lower vertebrates, but is not normally exhibited in mammals. We recently found that a subpopulation of human RPE cells can be stimulated in culture to generate multipotent self-renewing cells-the RPE stem cell (RPESC). RPESC can be expanded to generate RPE progeny that are a potential source for cell replacement therapy. Alternatively, RPESC can produce mesenchymal progeny which serve as a disease model of epiretinal membrane formation. Yet another potential application of RPESCs is activation within the eye to awaken dormant endogenous repair.

Epithelial–mesenchymal transition induces similar metabolic alterations in two independent breast cancer cell lines

Epithelial–mesenchymal transition (EMT) induces invasive properties in epithelial tumors and promotes metastasis. Although EMT-mediated cellular and molecular changes are well understood, very little is known about EMT-induced metabolic changes. HER2-positive BT-474 breast cancer cells were induced to undergo a stable EMT using mammosphere culture, as previously described by us for the ERα-positive MCF-7 breast cancer cells. Two epithelial breast cancer cell lines (BT-474 and MCF-7) were compared to their respective EMT-derived mesenchymal progeny (BT-474EMT and MCF-7EMT) for changes in metabolic pathways including glycolysis, glycogen metabolism, anabolic pathways and gluconeogenesis. Both EMT-derived cells displayed enhanced aerobic glycolysis along with the overexpression of specific glucose transporters, lactate dehydrogenase isoforms, monocarboxylate transporters and glycogen phosphorylase isoform. In contrast, both EMT-derived cells suppressed the expression of crucial enzymes in anabolic pathways and gluconeogenesis. STAT3, a transcription factor involved in tumor initiation and progression, plays a role in the EMT-related changes in the expression of specific enzymes and transporters. This study provides a broad overview of similar metabolic changes induced by EMT in two independent breast cancer cell lines. These metabolic changes may provide novel therapeutic targets for metastatic breast cancer.

Adult Human RPE Can Be Activated into a Multipotent Stem Cell that Produces Mesenchymal Derivatives

The retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. It begins as a plastic tissue, capable, in some species, of generating lens and retina, but differentiates early in development and remains normally nonproliferative throughout life. Here we show that a subpopulation of adult human RPE cells can be activated in vitro to a self-renewing cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers, proliferates extensively, and can redifferentiate into stable cobblestone RPE monolayers. Clonal studies demonstrate that RPESCs are multipotent and in defined conditions can generate both neural and mesenchymal progeny. This plasticity may explain human pathologies in which mesenchymal fates are seen in the eye, for example in proliferative vitroretinopathy (PVR) and phthisis bulbi. This study establishes the RPESC as an accessible, human CNS-derived multipotent stem cell, useful for the study of fate choice, replacement therapy, and disease modeling.

Abstract 4308: Dynamic regulation of CD24 and the invasive, CD44posCD24neg phenotype in breast cancer cell lines

Abstract Introduction The invasive, mesenchymal phenotype of CD44posCD24neg breast cancer cells has made them a promising target for eliminating the metastatic capacity of primary tumors. It has been previously demonstrated that CD44neg/lowCD24pos breast cancer cells lack the ability to give rise to their invasive CD44posCD24neg counterpart. Here we demonstrate that noninvasive, epithelial-like CD44posCD24pos cells readily give rise to invasive, mesenchymal CD44posCD24neg progeny in vivo and in vitro. This interconversion was found to be dependent upon Activin/Nodal signaling. Methods Breast cancer cell lines were sorted into CD44posCD24pos and CD44posCD24neg populations to evaluate their progeny for the expression of CD44, CD24, and markers of a mesenchymal phenotype. The FACS sorted populations were injected into immunocompromised mice to evaluate their tumorigenicity and invasiveness of the resulting xenografts. Results CD24 expression was dynamically regulated in vitro in all evaluated breast cancer cell lines. Furthermore, a single noninvasive, epithelial-like CD44posCD24pos cell had the ability to give rise to invasive, mesenchymal CD44posCD24neg progeny. Importantly, this interconversion occurred in vivo as CD44posCD24pos cells gave rise to xenografts with locally invasive borders as seen in xenografts initiated with CD44posCD24neg cells. Lastly, the ability of CD44posCD24pos cells to give rise to mesenchymal progeny, and vice versa, was blocked upon ablation of Activin/Nodal signaling. Conclusions Our data demonstrate that the invasive, mesenchymal CD44posCD24neg phenotype is under dynamic control in breast cancer cell lines both in vivo and in vitro. Furthermore, our observations suggest that targeted therapy against CD44posCD24neg tumor cells may have limited success in preventing primary tumor metastasis unless Activin/Nodal signaling is arrested. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4308.

Abstract 4239: Ectopic expression of EWS-FLI1 in human neural crest stem cells induces polycomb genes and maintenance of stemness

Abstract Ewing sarcoma family of tumors (ESFT) are aggressive bone and soft tissue tumors of unknown cellular origin. Most ESFT express EWS-FLI1, a chimeric protein which functions as a growth-promoting oncogene in ESFT but is toxic to most normal cells. A major difficulty in understanding EWS-FLI1 function has been the lack of an adequate model in which to study EWS-FLI1-induced transformation. Although the cell of origin of ESFT remains elusive, both mesenchymal (MSC) and neural crest (NCSC) have been implicated. We recently developed the tools to generate NCSC from human embryonic stem cells (hNCSC). In the current study we have used this model to test the hypothesis that NCSC are the cells of origin of ESFT and to evaluate the consequences of EWS-FLI1 expression on human NCSC biology. ESFT demonstrate variable degrees of neuro-mesenchymal differentiation potential. Similarly, we have found that hNCSC can be induced to differentiate into neural, glial and mesenchymal progeny. Significantly, Affymetrix whole genome expression profiling of 32 primary ESFT, 11 normal adult tissues, bone marrow-derived MSC and hNCSC revealed that ESFT are more similar to hNCSC than any other normal tissue, including MSC, thus further implicating NCSC in the origin of ESFT. To evaluate the consequences of EWS-FLI1 on these multipotent stem cells, hNCSC were stably transduced with an EWS-FLI1 lentivirus. Unlike most normal cells, hNCSC tolerated expression of the oncoprotein. Moreover, EWS-FLI1-transduced hNCSC continued to proliferate and maintain EWS-FLI1 expression in culture for several weeks after transduction. Affymetrix HuEx 1.0 expression profiling of hNCSC cells five days post-transduction with EWS-FLI1 demonstrated the expected induction and repression of well-established EWS-FLI1 targets and identified numerous other novel EWS-FLI1-regulated genes that are likely to be cell-type and situation specific. In contrast to control vector-transduced cells, EWS-FLI1 transduced hNCSC reproducibly maintained expression of the NCSC markers p75 and HNK-1, even after transfer to differentiation-inducing conditions. In addition, expression of the polycomb genes BMI1 and EZH2 was consistently and significantly upregulated in EWS-FLI1-transduced cells. In keeping with persistent expression of these stem cell-associated genes, EWS-FLI1-expressing cells retained the ability to form neurospheres upon transfer to non-adherent conditions and also the ability to differentiate into neural and mesenchymal lineages several weeks following transduction. In contrast, after six weeks in differentiation media, control cells had upregulated p16 and had undergone senescence. Together these data implicate NCSC in the origin of ESFT and suggest that EWS-FLI1 enables malignant transformation by inducing maintenance of their multipotent, stem cell state through deregulation of polycomb genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4239.

Enrichment for STRO‐1 expression enhances the cardiovascular paracrine activity of human bone marrow‐derived mesenchymal cell populations

The cardiovascular therapeutic potential of bone marrow mesenchymal stromal/stem cells (MSC) is largely mediated by paracrine effects. Traditional preparation of MSC has involved plastic adherence-isolation. In contrast, prospective immunoselection aims to improve cell isolation by enriching for mesenchymal precursor cells (MPC) at higher purity. This study compared the biological characteristics and cardiovascular trophic activity of plastic adherence-isolated MSC (PA-MSC) and MPC prepared from the same human donors by immunoselection for stromal precursor antigen-1 (STRO-1). Compared to PA-MSC, STRO-1-MPC displayed greater (1) clonogenicity, (2) proliferative capacity, (3) multilineage differentiation potential, and (4) mRNA expression of mesenchymal stem cell-related transcripts. In vitro assays demonstrated that conditioned medium from STRO-1-MPC had greater paracrine activity than PA-MSC, with respect to cardiac cell proliferation and migration and endothelial cell migration and tube formation. In keeping with this, STRO-1-MPC exhibited higher gene and protein expression of CXCL12 and HGF. Inhibition of these cytokines attenuated endothelial tube formation and cardiac cell proliferation, respectively. Paracrine responses were enhanced by using supernatant from STRO-1(Bright) MPC and diminished with STRO-1(Dim) conditioned medium. Together, these findings indicate that prospective isolation gives rise to mesenchymal progeny that maintain a higher proportion of immature precursor cells compared to traditional plastic adherence-isolation. Enrichment for STRO-1 is also accompanied by increased expression of cardiovascular-relevant cytokines and enhanced trophic activity. Immunoselection thus provides a strategy for improving the cardiovascular reparative potential of mesenchymal cells.

Dynamic regulation of CD24 and the invasive, CD44posCD24negphenotype in breast cancer cell lines

IntroductionThe invasive, mesenchymal phenotype of CD44posCD24neg breast cancer cells has made them a promising target for eliminating the metastatic capacity of primary tumors. It has been previously demonstrated that CD44neg/lowCD24pos breast cancer cells lack the ability to give rise to their invasive CD44posCD24neg counterpart. Here we demonstrate that noninvasive, epithelial-like CD44posCD24pos cells readily give rise to invasive, mesenchymal CD44posCD24neg progeny in vivo and in vitro. This interconversion was found to be dependent upon Activin/Nodal signaling.MethodsBreast cancer cell lines were sorted into CD44posCD24pos and CD44posCD24neg populations to evaluate their progeny for the expression of CD44, CD24, and markers of a mesenchymal phenotype. The populations, separated by fluorescence activated cell sorting (FACS) were injected into immunocompromised mice to evaluate their tumorigenicity and invasiveness of the resulting xenografts.ResultsCD24 expression was dynamically regulated in vitro in all evaluated breast cancer cell lines. Furthermore, a single noninvasive, epithelial-like CD44posCD24pos cell had the ability to give rise to invasive, mesenchymal CD44posCD24neg progeny. Importantly, this interconversion occurred in vivo as CD44posCD24pos cells gave rise to xenografts with locally invasive borders as seen in xenografts initiated with CD44posCD24neg cells. Lastly, the ability of CD44posCD24pos cells to give rise to mesenchymal progeny, and vice versa, was blocked upon ablation of Activin/Nodal signaling.ConclusionsOur data demonstrate that the invasive, mesenchymal CD44posCD24neg phenotype is under dynamic control in breast cancer cell lines both in vitro and in vivo. Furthermore, our observations suggest that therapies targeting CD44posCD24neg tumor cells may have limited success in preventing primary tumor metastasis unless Activin/Nodal signaling is arrested.

Budding Blood Cells

DEVELOPMENT Arguably the best-understood stem cells are those that generate the body's blood cells—hematopoietic stem cells (HSCs). Although the place of origin, the aortic-gonado-mesonephric (AGM) columns, is known, the specific cell type responsible for their emergence is not. By labeling cell populations in vivo either with the adhesion molecule VE cadherin or the transcription factor myocardin, Zovein et al. were able to follow endothelial and mesenchymal progeny, respectively. Their data point to an endothelial origin of HSCs for subsequent long-term, multilineage adult hematopoiesis. Further, the HSC endothelial progeny could be monitored as they traveled first to the fetal liver and then to the bone marrow for subsequent expansion and multilineage differentiation. Besides the AGM region, the authors also identified the placental vasculature and yolk sac as endothelial sources of HSCs. Hence, the origin of hematopoietic stem cells is endothelium, which itself comes from a transient mesenchymal population of cells. — BAP Cell Stem Cell 3 , 625 (2008).

A Novel Monoclonal Antibody (STRO-3) Identifies an Isoform of Tissue Nonspecific Alkaline Phosphatase Expressed by Multipotent Bone Marrow Stromal Stem Cells

Numerous studies support the concept that the nonhemopoietic cells of the bone marrow (BM), are derived from a population of multipotent bone marrow stromal stem cells (BMSSCs), which reside in perivascular niches within the bone marrow. These BMSSCs are thought to give rise not only to more cells that are phenotypically and functionally identical but also differentiated, lineage-committed mesenchymal progeny, including chondrocytes, smooth muscle cells, adipocytes, and osteoblasts. Recently, we have generated a novel monoclonal antibody (mAb) (designated STRO-3) that reacts with a minor subset of STRO-1(+) cells contained within adult BM aspirates and does not react with CD34(+) hemopoietic stem cells. Our results also show that STRO-3 identifies a high proportion of BMSSCs that possess extensive proliferative and multilineage differentiative capacity. Using retroviral expression cloning, we determined that STRO-3 binds to tissue nonspecific alkaline phosphatase (TNSALP), a cell-surface glycoprotein usually associated with cells of the osteoblast lineage. Studies presented here suggest that in addition to being expressed by osteoblasts, TNSALP may also represent a marker of immature BMSSCs in vivo. Finally, these studies suggest that antibodies to TNSALP may be used as an effective single marker of enrichment of BMSSCs from various tissues.

Skeletal myosphere-derived progenitor cell transplantation promotes neovascularization in δ-sarcoglycan knockdown cardiomyopathy

Bone marrow cells have been shown to contribute to neovascularization in ischemic hearts, whereas their impaired maturation to restore the δ-sarcoglycan (δ-SG) expression responsible for focal myocardial degeneration limits their utility to treat the pathogenesis of cardiomyopathy. Here, we report the isolation of multipotent progenitor cells from adult skeletal muscle, based on their ability to generate floating-myospheres. Myosphere-derived progenitor cells (MDPCs) are distinguishable from myogenic C2C12 cells and differentiate into vascular smooth muscle cells and mesenchymal progeny. The mutation in the δ-SG has been shown to develop vascular spasm to affect sarcolemma structure causing cardiomyopathy. We originally generated δ-SD knockdown (KD) mice and transplanted MDPCs into the hearts. MDPCs enhanced neoangiogenesis and restored δ-SG expression in impaired vasculatures through trans-differentiation, leading to improvement of cardiac function associated with paracrine effectors secretion. We propose that MDPCs may be the promising progenitor cells in skeletal muscle to treat δ-sarcoglycan complex mutant cardiomyopathy.

The co-expression of CD117 (c-kit) and osteocalcin in activated bone marrow stem cells in different diseases.

It is still difficult to identify a potential stem cell in the bone marrow which can give rise to haematopoiesis and mesenchymal cells. In the past, the stem cells for both tissues were considered to be from different stem cell pools, but it has been shown recently in vitro and in vivo that there is an unexpected plasticity at least among early haemopoietic progenitors which can give rise also to mesenchymal tissue. In an attempt to identify stem cells in the bone marrow, which are common precursors to both lineages, we observed that fibroblast-like periosteal cells changed their morphology towards an osteoblastic differentiation with a more cuboidal or triangular morphology especially close to metastatic infiltrates. As a marker for haemopoietic progenitors, we used an antibody against the tyrosine kinase receptor c-kit (CD117) and an anti-osteocalcin antibody to stain mesenchymal cells with a osteoblastic potential. Normal bone marrow specimens only showed a discrete expression pattern of CD117 and osteocalcin, but periosteal stem cells, which strongly co-express the applied haemopoietic and mesenchymal markers, were found particularly in the bone marrow of patients with infiltrates of malignant lymphoma or metastasis from prostate or breast cancer. The evaluation of bone marrow specimens from patients with an aplastic syndrome or myelodysplastic syndrome showed a more heterogenous expression pattern. Our results show that a stem cell candidate common to haematopoiesis and mesenchymal progeny can be detected in bone marrow specimens after activation, as demonstrated by the co-expression of CD117 and osteocalcin, which also seems to be associated with haematological diseases or metastatic infiltration in the bone marrow.