Human mesenchymal stem cells are thought to be multipotent cells, which are present in adult marrow, that can replicate as undifferentiated cells and that have the potential to differentiate to lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Cells that have the characteristics of human mesenchymal stem cells were isolated from marrow aspirates of volunteer donors. These cells displayed a stable phenotype and remained as a monolayer in vitro. These adult stem cells could be induced to differentiate exclusively into the adipocytic, chondrocytic, or osteocytic lineages. Individual stem cells were identified that, when expanded to colonies, retained their multilineage potential.

http://science.sciencemag.org/content/sci/284/5411/143.full.pdf

Multilineage Potential of Adult Human Mesenchymal Stem Cells

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

/pdf/human-adipose-tissue-is-a-source-of-multipotent-stem-cells-18jo72s52v.pdf

Human Adipose Tissue Is a Source of Multipotent Stem Cells

Marrow stromal cells can be isolated from other cells in marrow by their tendency to adhere to tissue culture plastic The cells have many of the characteristics of stem cells for tissues that can roughly be defined as mesenchymal, because they can be differentiated in culture into osteoblasts, chondrocytes, adipocytes, and even myoblasts Therefore, marrow stromal cells present an intriguing model for examining the differentiation of stem cells Also, they have several characteristics that make them potentially useful for cell and gene therapy

https://science.sciencemag.org/content/sci/276/5309/71.full.pdf

Marrow Stromal Cells as Stem Cells for Nonhematopoietic Tissues

Dentinal repair in the postnatal organism occurs through the activity of specialized cells, odontoblasts, that are thought to be maintained by an as yet undefined precursor population associated with pulp tissue. In this study, we isolated a clonogenic, rapidly proliferative population of cells from adult human dental pulp. These DPSCs were then compared with human bone marrow stromal cells (BMSCs), known precursors of osteoblasts. Although they share a similar immunophenotype in vitro, functional studies showed that DPSCs produced only sporadic, but densely calcified nodules, and did not form adipocytes, whereas BMSCs routinely calcified throughout the adherent cell layer with clusters of lipid-laden adipocytes. When DPSCs were transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue. In contrast, BMSCs formed lamellar bone containing osteocytes and surface-lining osteoblasts, surrounding a fibrous vascular tissue with active hematopoiesis and adipocytes. This study isolates postnatal human DPSCs that have the ability to form a dentin/pulp-like complex.

/pdf/postnatal-human-dental-pulp-stem-cells-dpscs-in-vitro-and-in-120dw1g1sy.pdf

Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo

Adult marrow-derived Mesenchymal Stem Cells (MSCs) are capable of dividing and their progeny are further capable of differentiating into one of several mesenchymal phenotypes such as osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, and adipocytes. In addition, these MSCs secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities. These secreted bioactive factors suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate mitosis and differentiation of tissue-intrinsic reparative or stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of MSCs into repair tissue. Several studies which tested the use of MSCs in models of infarct (injured heart), stroke (brain), or meniscus regeneration models are reviewed within the context of MSC-mediated trophic effects in tissue repair.

/pdf/mesenchymal-stem-cells-as-trophic-mediators-1sjekxtzx0.pdf

Mesenchymal stem cells as trophic mediators.

Stromal colonies with fibroblastic morphology grown from rabbit marrow cells in culture supplemented with foetal calf serum. In this study the same marrow cells cultured with autologous rabbit plasma and hydrocortisone form colonies of a single lineage that express the adipocytic phenotype. A comparison of the potential for differentiation of cloned cell populations grown from fibroblastic and adipocytic colonies has been made using an in vivo diffusion chamber assay. The adipocytic colonies differentiated and grew to a limited size in medium with rabbit plasma and hydrocortisone, but attempts to isolate them and expand them in this medium failed. When the serum supplement was changed to foetal calf serum at day 10 the cells in the adipocytic colonies acquired a less differentiated morphology, there was a large increase in colony growth and cells were produced in sufficient numbers for the diffusion chamber assay. Thirty one fibroblastic colonies and twenty one adipocytic colonies were isolated either by limiting dilution or ring cloning and then expanded. Of these, eleven fibroblastic and eight adipocytic colonies provided enough cells (2 x 10(5) to 2 x 10(6] for implantation and culture in the chambers. Four of the eleven fibroblastic and three of the eight adipocytic colonies formed an osteogenic tissue in the chambers. It was concluded that cells that have differentiated in an adipocytic direction are able to revert to a more proliferative stage and subsequently to differentiate along the osteogenic pathway. Adipocytic and fibroblastic cells cultured in vitro from marrow have, with osteogenic cells, a common precursor in adult marrow.

Adipocytic cells cultured from marrow have osteogenic potential

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

Fibroblastic colonies, each of which is derived from a single precursor cell (CFU-F), are formed when suspensions of marrow cells are cultured in vitro. The ability of marrow CFU-F to differentiate in vitro was investigated using the expression of alkaline phosphatase activity as a marker for osteogenic differentiation. In cultures of rabbit marrow cells the colonies formed varied in size, morphology and expression of enzyme activity, indicating that marrow stromal CFU-F are a heterogeneous population. Growth and differentiation of marrow CFU-F can be modified in vitro. Epidermal growth factor increased average colony size and reduced clonal expression of alkaline phosphatase activity to very low levels. Hydrocortisone activated the osteogenic differentiation programme within the cellular progeny of a wide spectrum of CFU-F. The results support the possible development of in vitro clonal methods for the study of differentiation and regulation of the osteogenic and other fibroblastic cell lines of the marrow stromal system.

Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F

WE have shown previously that a glycoprotein constituent of calcified cortical bone matrix is immunochemically identical to a component of blood plasma1,2. In bone and dentine this specific α-glycoprotein is concentrated in amounts relative to plasma albumin but is not present at greater concentrations than in plasma in various other tissues. This material, although mainly present in extra-vascular sites in bone, could originate from the plasma because it represents a substantial proportion of the radioactivity in bone tissue 12 d after injection of 14C-total plasma glycoprotein1. Rabbit bone α-glycoprotein is analogous to human α2HS-glycoprotein3,4, which is located in mineralising areas in normal human bone matrix5. To our knowledge direct conclusive proof of the tissue origin of the plasma α2HS-glycoprotein is lacking although most of the serum glycoproteins seem to be synthesised by the liver6,7. Nevertheless, its increased concentration in calcified tissues suggests that its presence in plasma could be a result of synthesis and release by bone tissue. We now report that the α2HS-glycoprotein is synthesised by the liver and a proportion is subsequently accumulated in bone tissue.

Origin of plasma alpha2HS-glycoprotein and its accumulation in bone.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780231002

M. E. Owen

Papers

Adipocytic cells cultured from marrow have osteogenic potential

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures

Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F

Origin of plasma alpha2HS-glycoprotein and its accumulation in bone.

The origin of bone cells in the postnatal organism