Query: Bone Marrow-Derived Mesenchymal Stem Cells Express the Pericyte Marker 3G5 in Culture and Show Enhanced Chondrogenesis in Hypoxic Conditions

title: Bone marrow derived mesenchymal stem cells express the pericyte marker 3G5 in culture and show enhanced chondrogenesis in hypoxic conditions
abstract: Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow derived stem cells.

title: Human infrapatellar fat pad derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor 2
abstract: IntroductionInfrapatellar fat pad (IPFP) is a possible source of stem cells for the repair of articular cartilage defects. In this study, adherent proliferative cells were isolated from digests of IPFP tissue. The effects of the expansion of these cells in fibroblast growth factor 2 (FGF 2) were tested on their proliferation, characterisation, and chondrogenic potential.MethodsIPFP tissue was obtained from six patients undergoing total knee replacement, and sections were stained with 3G5, alpha smooth muscle actin, and von Willebrand factor to identify different cell types in the vasculature. Cells were isolated from IPFP, and both mixed populations and clonal lines derived from them were characterised for cell surface epitopes, including 3G5. Cells were expanded with and without FGF 2 and were tested for chondrogenic differentiation in cell aggregate cultures.Results3G5 positive cells were present in perivascular regions in tissue sections of the IPFP, and proliferative adherent cells isolated from the IPFP were also 3G5 positive. However, 3G5 expression was on only a small proportion of cells in all populations and at all passages, including the clonally expanded cells. The cells showed cell surface epitope expression similar to adult stem cells. They stained strongly for CD13, CD29, CD44, CD90, and CD105 and were negative for CD34 and CD56 but were also negative for LNGFR (low affinity nerve growth factor receptor) and STRO1. The IPFP derived cells showed chondrogenic differentiation in cell aggregate cultures, and prior expansion with FGF 2 enhanced chondrogenesis. Expansion in FGF 2 resulted in greater downregulation of many cartilage associated genes, but on subsequent chondrogenic differentiation, they showed stronger upregulation of these genes and this resulted in greater matrix production per cell.ConclusionThese results show that these cells express mesenchymal stem cell markers, but further work is needed to determine the true origin of these cells. These results suggest that the expansion of these cells with FGF 2 has important consequences for facilitating their chondrogenic differentiation.

title: Mesenchymal and haematopoietic stem cells form a unique bone marrow niche
abstract: The cellular constituents forming the haematopoietic stem cell (HSC) niche in the bone marrow are unclear, with studies implicating osteoblasts, endothelial and perivascular cells. Here we demonstrate that mesenchymal stem cells (MSCs) identified using nestin expression, constitute an essential HSC niche component. Nestin+ MSCs contain all the bone marrow colony forming unit fibroblastic activity and can be propagated as non adherent 'mesenspheres' that can self renew and expand in serial transplantations. Nestin+ MSCs are spatially associated with HSCs and adrenergic nerve fibres, and highly express HSC maintenance genes. These genes, and others triggering osteoblastic differentiation, are selectively downregulated during enforced HSC mobilization or b3 adrenoreceptor activation. Whereas parathormone administration doubles the number of bone marrow nestin+ cells and favours their osteoblastic differentiation, in vivo nestin+ cell depletion rapidly reduces HSC content in the bone marrow. Purified HSCs home near nestin+ MSCs in the bone marrow of lethally irradiated mice, whereas in vivo nestin+ cell depletion significantly reduces bone marrow homing of haematopoietic progenitors. These results uncover an unprecedented partnership between two distinct somatic stem cell types and are indicative of a unique niche in the bone marrow made of heterotypic stem cell pairs.

title: Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell niche.
abstract: We review the morphofunctional characteristics of pericytes and report our observations. After a brief historical background, we consider the following aspects of pericytes: A) Origin in embryonic vasculogenesis (mesenchymal stem cells, neurocrest and other possible sources) and in embryonic and postnatal life angiogenesis (pre existing pericytes, fibroblast/ myofibroblasts and circulating progenitor cells) B) Location in pericytic microvasculature and in the other blood vessels (including transitional cell forms and absence in lymphatic vessels) incidence (differences depending on species, topographical location, and type and stage of vessels) and distribution (specific polarities) in blood vessels. C) Morphology (cell body, and longitudinal and circumferential cytoplasmic processes) structure (nucleus, cytoplasmic organelles and distribution of microtubules, intermediate filaments and microfilaments) and surface (caveolae system) D) Basement membrane disposition, formation, components and functions. E) Contacts with endothelial cells (ECs) (peg and socket arrangements, adherent junctions and gap junctions) and with basal membrane (adhesion plaques) F) Molecular expression (pericyte marker identification) G) Functions, such as vessel stabilization, regulation of vascular tone and maintenance of local and tissue homeostasis (contractile capacity and vessel permeability regulation) matrix protein synthesis, macrophage like properties, immunological defense, intervention in coagulation, participation in mechanisms that regulate the quiescent and angiogenic stages of blood vessels (including the behaviour of pericytes during sprouting angiogenesis and intussuceptive vascular growth, as well as pericyte interactions with endothelium and other cells, and with extracellular matrix) and plasticity, as progenitor cells with great mesenchymal potential, originating other pericytes, fibroblast/myofibroblasts, preadipocytes, chondroblasts, osteoblasts, odontoblasts, vascular smooth muscle and myointimal cells. This mesenchymal capacity is seen in a broad section on the perivascular mesenchymal cell niche hypothesis and in the concept of pericyte and EC "marriage and divorce" H) Peculiar pericyte types, such as hepatic stellate cells (Ito cells) bone marrow reticular cells and mesangial cells. I) Involvement in pathological processes, such as repair through granulation tissue, pericyte derived tumors, tumor angiogenesis and tumoral cell metastasis, diabetic microangiopathy, fibrosis, atherosclerosis and calcific vasculopathy, lymphedema distichiasis, chronic venous insufficiency, pulmonary hypertension, Alzheimer disease and multiple sclerosis.  Clinical and therapeutic implications (de stabilization of vessels or formation of a stable vasculature)

title: A perivascular origin for mesenchymal stem cells in multiple human organs.
abstract: Mesenchymal stem cells (MSCs) the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

title: Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources: Their Biology and Role in Regenerative Medicine
abstract: To date, 5 different human dental stem/progenitor cells have been isolated and characterized: dental pulp stem cells (DPSCs) stem cells from exfoliated deciduous teeth (SHED) periodontal ligament stem cells (PDLSCs) stem cells from apical papilla (SCAP) and dental follicle progenitor cells (DFPCs) These postnatal populations have mesenchymal stem cell like (MSC) qualities, including the capacity for self renewal and multilineage differentiation potential. MSCs derived from bone marrow (BMMSCs) are capable of giving rise to various lineages of cells, such as osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic cells. The dental tissue derived stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells. However, they also have the ability to give rise to other cell lineages similar to, but different in potency from, that of BMMSCs. This article will review the isolation and characterization of the properties of different dental MSC like populations in comparison with those of other MSCs, such as BMMSCs. Important issues in stem cell biology, such as stem cell niche, homing, and immunoregulation, will also be discussed.

title: Immunophenotype of Human Adipose Derived Cells: Temporal Changes in Stromal Associated and Stem Cell Associated Markers
abstract: Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue derived stromal vascular fraction (SVF) cells relative to serial passaged adipose derived stem cells (ASCs) The initial SVF cells contained colony forming unit fibroblasts at a frequency of 1:32. Colony forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively) The immunophenotype of the adipose derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell associated marker CD34 was at peak levels in the SVF cells and/or early passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug resistance transport protein (ABCG2) both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell associated markers (CD31, CD144 or VE cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose derived cells in fetal bovine serum supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.

title: Mesenchymal stem cells in health and disease
abstract: Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues. They can differentiate into cells of the mesodermal lineage, such as adipocytes, osteocytes and chondrocytes, as well as cells of other embryonic lineages. MSCs can interact with cells of both the innate and adaptive immune systems, leading to the modulation of several effector functions. After in vivo administration, MSCs induce peripheral tolerance and migrate to injured tissues, where they can inhibit the release of pro inflammatory cytokines and promote the survival of damaged cells. This Review discusses the targets and mechanisms of MSC mediated immunomodulation and the possible translation of MSCs to new therapeutic approaches.

title: SSEA 4 identifies mesenchymal stem cells from bone marrow.
abstract: Adult bone marrow (BM) contains hematopoietic stem cells (HSCs) as well as a nonhematopoietic, stromal cell population. Within this stromal population are mesenchymal stem cells (MSCs) which not only support hematopoiesis but also differentiate into multiple lineages, including fat, bone, and cartilage. Because of this multipotentiality, the MSC is an attractive candidate for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, research progress has been hampered by the limited existing knowledge of the biology of these cells, particularly by the lack of a suitable marker for their prospective isolation. Here, we report that SSEA 4, an early embryonic glycolipid antigen commonly used as a marker for undifferentiated pluripotent human embryonic stem cells and cleavage to blastocyst stage embryos, also identifies the adult mesenchymal stem cell population.

title: Hypoxic condition enhances chondrogenesis in synovium derived mesenchymal stem cells
abstract: BackgroundThe chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors, including oxygen tensions, growth factors, and cytokines. Evidences have suggested that low oxygen tension seems to be an important regulatory factor in the proliferation and chondrogenic differentiation in various MSCs. Recent studies report that synovium derived mesenchymal stem cells (SDSCs) are a potential source of stem cells for the repair of articular cartilage defects. But, the effect of low oxygen tension on the proliferation and chondrogenic differentiation in SDSCs has not characterized. In this study, we investigated the effects of hypoxia on proliferation and chondrogenesis in SDSCs.MethodSDSCs were isolated from patients with osteoarthritis at total knee replacement. To determine the effect of oxygen tension on proliferation and colony forming characteristics of SDSCs, A colony forming unit (CFU) assay and cell counting based proliferation assay were performed under normoxic (21% oxygen) or hypoxic (5% oxygen) For in vitro chondrogenic differentiation, SDSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis. qRT PCR, histological assay, and glycosoaminoglycan assays were determined to assess chondrogenesis.ResultsLow oxygen condition significantly increased proliferation and colony forming characteristics of SDSCs compared to that of SDSCs under normoxic culture. Similar pellet size and weight were found for chondrogensis period under hypoxia and normoxia condition. The mRNA expression of types II collagen, aggrecan, and the transcription factor SOX9 was increased under hypoxia condition. Histological sections stained with Safranin O demonstrated that hypoxic conditions had increased proteoglycan synthesis. Immunohistochemistry for types II collagen demonstrated that hypoxic culture of SDSCs increased type II collagen expression. In addition, GAG deposition was significantly higher in hypoxia compared with normoxia at 21 days of differentiation.ConclusionThese findings show that hypoxia condition has an important role in regulating the synthesis ECM matrix by SDSCs as they undergo chondrogenesis. This has important implications for cartilage tissue engineering applications of SDSCs.