Identity and ranking of colonic mesenchymal stromal cells

Although ongoing clinical trials utilize systemic administration of bone-marrow mesenchymal stromal cells (BM-MSCs) in Crohn's disease (CD), nothing is known about the presence and the function of mesenchymal stromal cells (MSCs) in the normal human bowel. MSCs are bone marrow (BM) multipotent cells supporting hematopoiesis with the potential to differentiate into multiple skeletal phenotypes. A recently identified new marker, CD146, allowing to prospectively isolate MSCs from BM, renders also possible their identification in different tissues. In order to elucidate the presence and functional role of MSCs in human bowel we analyzed normal adult colon sections and isolated MSCs from them. In colon (C) sections, resident MSCs form a net enveloping crypts in lamina propria, coinciding with structural myofibroblasts or interstitial stromal cells. Nine sub-clonal CD146(+) MSC lines were derived and characterized from colon biopsies, in addition to MSC lines from five other human tissues. In spite of a phenotype qualitative identity between the BM- and C-MSC populations, they were discriminated and categorized. Similarities between C-MSC and BM-MSCs are represented by: Osteogenic differentiation, hematopoietic supporting activity, immune-modulation, and surface-antigen qualitative expression. The differences between these populations are: C-MSCs mean intensity expression is lower for CD13, CD29, and CD49c surface-antigens, proliferative rate faster, life-span shorter, chondrogenic differentiation rare, and adipogenic differentiation completely blocked. Briefly, BM-MSCs, deserve the rank of progenitors, whereas C-MSCs belong to the restricted precursor hierarchy. The presence and functional role of MSCs in human colon provide a rationale for BM-MSC replacement therapy in CD, where resident bowel MSCs might be exhausted or diverted from their physiological functions.     

Regulation of pancreatic function by connective tissue growth factor (CTGF,CCN2)

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich matricellular secreted protein that regulates diverse cell functions including adhesion, migration, proliferation, differentiation, survival, senescence and apoptosis. In the pancreas, CTGF/CCN2 regulates critical functions including β cell replication during embryogenesis, stimulation of fibrogenic pathways in pancreatic stellate cells during pancreatitis, and regulation of the epithelial and stromal components in pancreatic ductal adenocarcinoma. This article reviews the evidence establishing CTGF/CCN2 as an important player in pancreatic physiology and pathology, highlighting the specific cell types that are involved in each process and the importance of CTGF/CCN2 as a component of autocrine or paracrine signaling within or between these various cells. Translational applications, including the potential for CTGF/CCN2-based therapies in diabetes, fibrosis, or cancer, are discussed.     

Regulation and function of connective tissue growth factor/CCN2 in tissue repair, scarring and fibrosis

Connective tissue growth factor (CTGF, CCN2) is a secreted protein with major roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. It is a member of the CCN family of immediate-early gene products which are characterised by four discrete protein modules in which reside growth factor binding domains, functional motifs for integrin recognition, heparin and proteoglycan binding, and dimerization motifs. A primary function of CTGF is to modulate and coordinate signaling responses involving cell surface proteoglycans, key components of the extracellular matrix, and growth factors. Integration of these molecular cues regulates growth factor and receptor interactions, cell motility and mesenchymal cell activation and differentiation in tissue remodelling. Abnormal amplification of CTGF dependent signals results in a failure to terminate tissue repair, leading pathological scarring in conditions such as fibrosis and cancer.     

Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) Regulates Hematopoiesis and Bone Formation In Vivo

Background

Tissue inhibitor of metalloproteinases-3 (TIMP-3) inhibits matrix metalloproteinases and membrane-bound sheddases. TIMP-3 is associated with the extracellular matrix and is expressed in highly remodeling tissues. TIMP-3 function in the hematopoietic system is unknown.

Methodology/Principal Findings

We now report that TIMP-3 is highly expressed in the endosteal region of the bone marrow (BM), particularly by osteoblasts, endothelial and multipotent mesenchymal stromal cells which are all important cellular components of hematopoietic stem cell (HSC) niches, whereas its expression is very low in mature leukocytes and hematopoietic stem and progenitor cells. A possible role of TIMP-3 as an important niche component was further suggested by its down-regulation during granulocyte colony-stimulating factor-induced mobilization. To further investigate TIMP-3 function, mouse HSC were retrovirally transduced with human TIMP-3 and transplanted into lethally irradiated recipients. TIMP-3 overexpression resulted in decreased frequency of B and T lymphocytes and increased frequency of myeloid cells in blood and BM, increased Lineage-negative Sca-1⁺KIT⁺ cell proliferation in vivo and in vitro and increased colony-forming cell trafficking to blood and spleen. Finally, over-expression of human TIMP-3 caused a late onset fatal osteosclerosis.

Conclusions/Significance

Our results suggest that TIMP-3 regulates HSC proliferation, differentiation and trafficking in vivo, as well as bone and bone turn-over, and that TIMP-3 is expressed by stromal cells forming HSC niches within the BM. Thus, TIMP-3 may be an important HSC niche component regulating both hematopoiesis and bone remodeling.     

A role for Lys-His-Gly-NH2 in avian and murine B cell development

Lys-His-Gly-NH2 has been claimed to selectively induce B cell precursors to differentiate into mature B lymphocytes. In the present study, the effects of this tripeptide and a control compound having the reverse sequence (Gly-His-Lys-NH2) on growth and differentiation of chicken and mouse B cell precursors were investigated. When chicken bone marrow (BM) cells from 15-day-old embryos were treated for 18 hr with either of the tripeptides, the frequency of Bu-1 antigen-bearing cells increased. Moreover, when embryonic bursa cells were stimulated in vitro with phorbol myristate acetate, which induces them to proliferate and undergo terminal differentiation into immunoglobulin (Ig)-secreting cells, these compounds caused a 10-fold increase in the number of Ig-secreting cells but did not increase cell proliferation. They had no effect on neonatal or adult bursa cells. Embryonic bursa cells were cultured in the presence of either of the tripeptides and metabolically labeled with [35S]methionine. When immunoprecipitated Ig was analyzed by two-dimensional gel electrophoresis, no differences in mu heavy or lambda light chain diversity patterns could be detected, indicating that neither of these compounds enhances Ig diversification. The effect of these tripeptides on murine B cell precursors was assayed in cultures of BM cells depleted of mature B cells by 5-fluorouracil. When precursor cells were incubated without adherent BM stromal cells, they did not respond to the tripeptides. However, after incubation of precursors with adherent stromal BM cells for 2 days, followed by treatment with either of the two tripeptides, differentiation into lipopolysaccharide-reactive mature B cells took place. Incubation of precursors with adherent stromal BM cells in the absence of tripeptides was not sufficient to allow the precursors to complete differentiation. In addition, both tripeptides acted synergistically with interleukin 1 or interleukin 3. In conclusion, these tripeptides seem to enhance precursor B cell differentiation in a lineage-nonspecific manner rather than to function as lineage-specific differentiation hormones.     

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF‐MSCs are less prone to senescence with respect to BM‐MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF‐MSCs are able to return to the basal condition more efficiently with respect to BM‐MSCs. Indeed, AF‐MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF‐MSCs may represent a valid alternative to BM‐MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF‐MSCs and BM‐MSCs may pave the way to their rational use in the medical field.     

Mesenchymal stromal cell‐derived extracellular vesicles as cell‐free biologics for the ex vivo expansion of hematopoietic stem cells

Hematopoietic stem cell transplantation (HSCT) is the ultimate choice of treatment for patients with hematological diseases and cancer. The success of HSCT is critically dependent on the number and engraftment efficiency of the transplanted donor hematopoietic stem cells (HSCs). Various studies show that bone marrow‐derived mesenchymal stromal cells (MSCs) support hematopoiesis and also promote ex vivo expansion of HSCs. MSCs exert their therapeutic effect through paracrine activity, partially mediated through extracellular vesicles (EVs). Although the physiological function of EVs is not fully understood, inspiring findings indicate that MSC‐derived EVs can reiterate the hematopoiesis, supporting the ability of MSCs by transferring their cargo containing proteins, lipids, and nucleic acids to the HSCs. The activation state of the MSCs or the signaling mechanism that prevails in them also defines the composition of their EVs, thereby influencing the fate of HSCs. Modulating or preconditioning MSCs to achieve a specific composition of the EV cargo for the ex vivo expansion of HSCs is, therefore, a promising strategy that can overcome several challenges associated with the use of naïve/unprimed MSCs. This review aims to speculate upon the potential role of preconditioned/primed MSC‐derived EVs as “cell‐free biologics,” as a novel strategy for expanding HSCs in vitro.     

Specific signals involved in the long-term maintenance of radiation-induced fibrogenic differentiation: a role for CCN2 and low concentration of TGF-beta1

The fibrogenic differentiation of resident mesenchymal cells is a key parameter in the pathogenesis of radiation fibrosis and is triggered by the profibrotic growth factors transforming growth factor (TGF)-beta1 and CCN2. TGF-beta1 is considered the primary inducer of fibrogenic differentiation and is thought to control its long-term maintenance, whereas CCN2 is considered secondary effector of TGF-beta1. Yet, in long-term established fibrosis like that associated with delayed radiation enteropathy, in situ TGF-beta1 deposition is low, whereas CCN2 expression is high. To explore this apparent paradox, cell response to increasing doses of TGF-beta1 was investigated in cells modeling initiation and maintenance of fibrosis, i.e., normal and fibrosis-derived smooth muscle cells, respectively. Activation of cell-specific signaling pathways by low TGF-beta1 doses was demonstrated with a main activation of the Rho/ROCK pathway in fibrosis-derived cells, whereas the Smad pathway was mainly activated in normal cells. This leads to subsequent and cell-specific regulation of the CCN2 gene. These results suggested a specific profibrotic role of CCN2 in fibrosis-initiated cells. Furthermore, the modulation of CCN2 expression by itself and the combination of TGF-beta1 and CCN2 was investigated in fibrosis-derived cells. In fibrosis-initiated cells CCN2 triggered its autoinduction; furthermore, low concentration of TGF-beta1-potentiated CCN2 autoinduction. Our findings showed a differential requirement and action of TGF-beta1 in the fibrogenic response of normal vs. fibrosis-derived cells. This study defines a novel Rho/ROCK but Smad3-independent mode of TGF-beta signaling that may operate during the chronic stages of fibrosis and provides evidence of both specific and combinatorial roles of low TGF-beta1 dose and CCN2.     

Oncostatin M Maintains the Hematopoietic Microenvironment in the Bone Marrow by Modulating Adipogenesis and Osteogenesis

The bone marrow (BM) is an essential organ for hematopoiesis in adult, in which proliferation and differentiation of hematopoietic stem/progenitor cells (HSPC) is orchestrated by various stromal cells. Alterations of BM hematopoietic environment lead to various hematopoietic disorders as exemplified by the linking of fatty marrow with increased adipogenesis to anemia or pancytopenia. Therefore, the composition of mesenchymal stromal cell (MSC)-derived cells in the BM could be crucial for proper hematopoiesis, but the mechanisms underlying the MSC differentiation for hematopoiesis remain poorly understood. In this study, we show that Oncostatin M (OSM) knock out mice exhibited pancytopenia advancing fatty marrow with age. OSM strongly inhibited adipogenesis from BM MSC in vitro, whereas it enhanced their osteogenesis but suppressed the terminal differentiation. Intriguingly, OSM allowed the MSC-derived cells to support the ex vivo expansion of HSPC effectively as feeder cells. Furthermore, the administration of OSM in lethally irradiated wild-type mice blocked fatty marrow and enhanced the recovery of HSPC number in the BM and peripheral blood cells after engraftment of HSPC. Collectively, OSM plays multiple critical roles in the maintenance and development of the hematopoietic microenvironment in the BM at a steady state as well as after injury.     

Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.     

CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis

CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.     

CCN2, connective tissue growth factor, stimulates collagen deposition by gingival fibroblasts via module 3 and alpha6- and beta1 integrins

CCN2, (connective tissue growth factor, CTGF) is a matricellular factor associated with fibrosis that plays an important role in the production and maintenance of fibrotic lesions. Increased collagen deposition and accumulation is a common feature of fibrotic tissues. The mechanisms by which CCN2/CTGF contributes to fibrosis are not well understood. Previous studies suggest that CTGF exerts some of its biological effects at least in part by integrin binding, though this mechanism has not been previously shown to contribute to fibrosis. Utilizing full length CCN2/CTGF, CCN2/CTGF fragments, and integrin neutralizing antibodies, we provide evidence that the effects of CCN2/CTGF to stimulate extracellular matrix deposition by gingival fibroblasts are mediated by the C-terminal half of CCN2/CTGF, and by alpha6 and beta1 integrins. In addition, a synthetic peptide corresponding to a region of CCN2/CTGF domain 3 that binds alpha6beta1 inhibits the collagen-deposition assay. These studies employed a new and relatively rapid assay for CCN2/CTGF-stimulated collagen deposition based on Sirius Red staining of cell layers. Data obtained support a pathway in which CCN2/CTGF could bind to alpha6beta1 integrin and stimulate collagen deposition. These findings provide new experimental methodologies applicable to uncovering the mechanism and signal transduction pathways of CCN2/CTGF-mediated collagen deposition, and may provide insights into potential therapeutic strategies to treat gingival fibrosis and other fibrotic conditions.     

Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells

In recent years, a large number of studies have contributed to our understanding of the immunomodulatory mechanisms used by multipotent mesenchymal stem cells (MSCs). Initially isolated from the bone marrow (BM), MSCs have been found in many tissues but the strong immunomodulatory properties are best studied in BM MSCs. The immunomodulatory effects of BM MSCs are wide, extending to T lymphocytes and dendritic cells, and are therapeutically useful for treatment of immune-related diseases including graft-versus-host disease as well as possibly autoimmune diseases. However, BM MSCs are very rare cells and require an invasive procedure for procurement. Recently, MSCs have also been found in fetal-stage embryo-proper and extra-embryonic tissues, and these human fetal MSCs (F-MSCs) have a higher proliferative profile, and are capable of multilineage differentiation as well as exert strong immunomodulatory effects. As such, these F-MSCs can be viewed as alternative sources of MSCs. We review here the current understanding of the mechanisms behind the immunomodulatory properties of BM MSCs and F-MSCs. An increase in our understanding of MSC suppressor mechanisms will offer insights for prevalent clinical use of these versatile adult stem cells in the near future.     

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-β1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-β1. Conversely, TGF-β1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-β1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.     

CCN1 and CCN2: blood brothers in angiogenic action

CCN2/connective tissue growth factor (CTGF) is a matricellular protein essential for skeletal development during embryogenesis. In adulthood, aberrant CCN2 expression is associated with many malignancies and fibrosis of virtually every organ. Despite its prominent expression in endothelial cells in the vasculature, the role of CCN2 in vessel development was unknown. In a recent study, Hall-Glenn et al. (PLoS ONE 7:e30562) have revealed the role of CCN2 in developmental angiogenesis through a detailed analysis of how CCN2 mediates the interaction between vascular endothelial cells and pericytes. In addition, CCN2 also regulates endothelial basement membrane formation during vessel formation. Here I compare the angiogenic activities of CCN2 during embryogenesis to those of its homologous family member CCN1 (CYR61), which is essential for cardiovascular development. Understanding the angiogenic actions of CCN1 and CCN2 may have implication in the development of therapeutic strategies targeting these proteins for the treatment of diseases such as cancer and fibrosis.     

CCN2 (connective tissue growth factor) promotes fibroblast adhesion to fibronectin

In vivo, CCN2 (connective tissue growth factor) promotes angiogenesis, osteogenesis, tissue repair, and fibrosis, through largely unknown mechanisms. In vitro, CCN2 promotes cell adhesion in a variety of systems via integrins and heparin sulfate proteoglycans (HSPGs). However, the physiological relevance of CCN2-mediated cell adhesion is unknown. Here, we find that HSPGs and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade are required for adult human dermal fibroblasts to adhere to CCN2. Endogenous CCN2 directly binds fibronectin and the fibronectin receptors integrins alpha4 beta1 and alpha5 and syndecan 4. Using Ccn2-/- mouse embryonic fibroblasts, we show that loss of endogenous CCN2 results in impaired spreading on fibronectin, delayed alpha-smooth muscle actin stress fiber formation, and reduced ERK and focal adhesion kinase phosphorylation. These results suggest that a physiological role of CCN2 is to potentiate the ability of fibroblasts to spread on fibronectin, which may be important in modulating fibroblast adhesion to the provisional matrix during tissue development and wound healing. These results are consistent with the notion that a principal function of CCN2 is to modulate receptor/ligand interactions in vivo.