Objective
To compare the properties of human mesenchymal stem cells (MSCs) isolated from bone marrow, synovium, periosteum, skeletal muscle, and adipose tissue.

Methods
Human mesenchymal tissues were obtained from 8 donors during knee surgery for ligament injury. After collagenase digestion or gradient-density separation, nucleated cells were plated at an appropriate density for expansion at the maximum rate without colony-to-colony contact. Yield, expandability, differentiation potential, and epitope profile were compared among MSCs from the 5 different tissue sources.

Results
Colony number per 103 nucleated cells was lower, and cell number per colony was higher, in bone marrow than in other mesenchymal tissues. When the cells were replated at low density every 14 days, bone marrow–, synovium-, and periosteum-derived cells retained their proliferation ability even at passage 10. In chondrogenesis studies in which the cells were pelleted and cultured in vitro, pellets from bone marrow–, synovium-, and periosteum-derived cells were shown to be larger and stained more extensively for cartilage matrix. Synovium-derived cells, in particular, had the greatest ability for chondrogenesis. In adipogenesis experiments, the frequency of oil red O–positive colonies was highest in synovium- and adipose tissue–derived cells. In studies of osteogenesis, the rate of alizarin red–positive colonies was highest in bone marrow–, synovium-, and periosteum-derived cells. For epitope profiling, 15 surface antigens were measured. Most appeared to have similar epitope profiles irrespective of cell source.

Conclusion
Our findings indicate that there are significant differences in MSC properties according to tissue source, beyond donor and experimental variation. Superiority of synovium as a potential source of MSCs for clinical applications was demonstrated.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.21212

Comparison of human stem cells derived from various mesenchymal tissues: Superiority of synovium as a cell source

The identification of multipotential mesenchymal stem cells (MSCs) derived from adult human tissues, including bone marrow stroma and a number of connective tissues, has provided exciting prospects for cell-based tissue engineering and regeneration. This review focuses on the biology of MSCs, including their differentiation potentials in vitro and in vivo, and the application of MSCs in tissue engineering. Our current understanding of MSCs lags behind that of other stem cell types, such as hematopoietic stem cells. Future research should aim to define the cellular and molecular fingerprints of MSCs and elucidate their endogenous role(s) in normal and abnormal tissue functions.

/pdf/adult-mesenchymal-stem-cells-and-cell-based-tissue-57ivt8q31b.pdf

Adult mesenchymal stem cells and cell-based tissue engineering.

We report here the isolation of a population of non-transformed pluripotent human cells from bone marrow after a unique expansion/selection procedure. This procedure was designed to provide conditions resembling the in vivo microenvironment that is home for the most-primitive stem cells. Marrow-adherent and -nonadherent cells were co-cultured on fibronectin, at low oxygen tension, for 14 days. Colonies of small adherent cells were isolated and further expanded on fibronectin at low density, low oxygen tension with 2% fetal bovine serum. They expressed high levels of CD29, CD63, CD81, CD122, CD164, hepatocyte growth factor receptor (cMet), bone morphogenetic protein receptor 1B (BMPR1B), and neurotrophic tyrosine kinase receptor 3 (NTRK3) and were negative for CD34, CD36, CD45, CD117 (cKit) and HLADR. The embryonic stem cell markers Oct-4 and Rex-1, and telomerase were expressed in all cultures examined. Cell-doubling time was 36 to 72 hours, and cells have been expanded in culture for more than 50 population doublings. This population of cells was consistently isolated from men and women of ages ranging from 3- to 72-years old. Colonies of cells expressed numerous markers found among embryonic stem cells as well as mesodermal-, endodermal- and ectodermal-derived lineages. They have been differentiated to bone-forming osteoblasts, cartilage-forming chondrocytes, fat-forming adipocytes and neural cells and to attachment-independent spherical clusters expressing genes associated with pancreatic islets. Based on their unique characteristics and properties, we refer to them as human marrow-isolated adult multilineage inducible cells, or MIAMI cells. MIAMI cells proliferate extensively without evidence of senescence or loss of differentiation potential and thus may represent an ideal candidate for cellular therapies of inherited or degenerative diseases.

/pdf/marrow-isolated-adult-multilineage-inducible-miami-cells-a-2o0cbxqnhx.pdf

Marrow-isolated adult multilineage inducible (MIAMI) cells, a unique population of postnatal young and old human cells with extensive expansion and differentiation potential.

Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large-scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fibroblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA-class I and induces low HLA-DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF-cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/pdf/10.1634/stemcells.2004-0331

Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are progenitors of all connective tissue cells. In adults of multiple vertebrate species, MSCs have been isolated from bone marrow (BM) and other tissues, expanded in culture, and differentiated into several tissue-forming cells such as bone, cartilage, fat, muscle, tendon, liver, kidney, heart, and even brain cells. Recent advances in the practical end of application of MSCs toward regeneration of a human-shaped articular condyle of the synovial joint is one example of their functionality and versatility. The present review not only outlines several approaches relevant to the isolation and therapeutic use of MSCs, but also presents several examples of phenotypic and functional characterization of isolated MSCs and their progeny.

Mesenchymal stem cells: isolation and therapeutics.

Bone marrow mesenchymal stem cells (MSCs) have the capacity for renewal and the potential to differentiate into multiple lineages of mesenchymal tissues. In the laboratory, MSCs have the tendency to adhere to culture dish plastic and are characterized by fibroblastic morphology, but possess no specific markers to select them. To isolate and purify MSCs from bone marrow, we use a culture device-a plastic culture dish comprising a plate with 3-microm pores-to sieve out a homogeneous population of cells (termed size-sieved [SS] cells) from bone marrow aspirates. SS cells that adhered to the upper porous plate surface were a relatively homogeneous population as indicated by morphology and other criteria, such as surface markers. They had the capacity for self-renewal and the multilineage potential to form bone, fat, and cartilage, and satisfy the characteristics of MSCs. In addition, if all the cells from each passage had been plated and cultured in our defined conditions, over 10(14) SS cells would have been obtained from each 10-ml aspirate in 15 additional weeks of culture. This technically simple method leads to an efficient isolation and purification of cells with the characteristics of MSCs.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/pdf/10.1634/stemcells.20-3-249

Isolation and characterization of size-sieved stem cells from human bone marrow

Maternal cells can become engrafted in various fetal organs during pregnancy. The nature of the cells and the mechanisms of maternofetal cell trafficking are not clear. We demonstrate that human lineage-negative, CD34-negative (Lin(-)CD34(-)) multipotent mesenchymal stromal cells express alpha(2), alpha(4), alpha(5), and beta(1) integrins, which mediate their adhesion to endothelium, and vascular endothelial growth factor receptor-1 (VEGFR-1), which mediates their response to vascular endothelial growth factor A (VEGF-A). A maternal-fetal VEGF-A concentration gradient exists across the placental barrier, and cord blood plasma induces transendothelial and trans-Matrigel migration of stem cells in vitro. Migration is inhibited by a VEGF-A-neutralizing antibody or antibodies against VEGFR-1 or integrin alpha(2), alpha(4), alpha(5), or beta(1). When Lin(-)CD34(-) multipotent mesenchymal stromal cells are transferred to rat maternal venous blood, they traffic through the placenta, engraft in various fetal organs, and persist in offspring for at least 12 weeks. Cell proliferation ability is retained in the xenogeneic placenta. Maternofetal trafficking is significantly reduced by blocking antibodies against integrins alpha(2), alpha(4), alpha(5), and beta(1) or VEGFR-1. These results suggest that maternal microchimerism arises by the trafficking of multipotent mesenchymal stromal cells via VEGF-A- and integrin-dependent pathways across the hemochorial placenta to fetal tissues.

https://stemcellsjournals.onlinelibrary.wiley.com/doi/pdf/10.1634/stemcells.2007-0406

Trafficking of multipotent mesenchymal stromal cells from maternal circulation through the placenta involves vascular endothelial growth factor receptor-1 and integrins.

Hung Li

Papers

Isolation and characterization of size-sieved stem cells from human bone marrow

Trafficking of multipotent mesenchymal stromal cells from maternal circulation through the placenta involves vascular endothelial growth factor receptor-1 and integrins.