Placental tissue draws great interest as a source of cells for regenerative medicine because of the phenotypic plasticity of many of the cell types isolated from this tissue. Furthermore, placenta, ...which is involved in maintaining fetal tolerance, contains cells that display immunomodulatory properties. These two features could prove useful for future cell therapy‐based clinical applications. Placental tissue is readily available and easily procured without invasive procedures, and its use does not elicit ethical debate. Numerous reports describing stem cells from different parts of the placenta, using nearly as numerous isolation and characterization procedures, have been published. Considering the complexity of the placenta, an urgent need exists to define, as clearly as possible, the region of origin and methods of isolation of cells derived from this tissue. On March 23–24, 2007, the first international Workshop on Placenta Derived Stem Cells was held in Brescia, Italy. Most of the research published in this area focuses on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. The aim of this review is to summarize and provide the state of the art of research in this field, addressing aspects such as cell isolation protocols and characteristics of these cells, as well as providing preliminary indications of the possibilities for use of these cells in future clinical applications.
Disclosure of potential conflicts of interest is found at the end of this article.
Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological ...cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age‐ or OA etiology‐dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA‐etiology groups (age‐related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase‐polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor‐site morbidity, typically containing >107 mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible.
Disclosure of potential conflicts of interest is found at the end of this article.
Coexpression of CD140b (PDGFRβ) and CD146 has been used to isolate endometrial mesenchymal stem-like cells (eMSCs), which have a perivascular location. This study aims to evaluate a single marker for ...purifying eMSCs. Using an antibody panel with novel specificities, we screened human endometrial tissues and stromal cell suspensions by flow cytometry and immunohistochemistry to identify perivascular markers. Sorted subpopulations were examined for colony-forming unit (CFU), self-renewal, and differentiation assays for mesenchymal stem cell (MSC) function. We also transplanted sorted eMSCs under the kidney capsule of superimmunodeficient NSG mice. Magnetic bead selection was compared with flow cytometry sorting (flow sorting) using CFU assay. One novel marker (W5C5) was particularly effective in selecting eMSCs. W5C5+ cells comprise 4.2 ± 0.6% (n = 34) of endometrial stromal cells and reside predominantly in a perivascular location in both basal and functional layers of endometrium. The clonogenicity of W5C5+ cells is significantly greater than W5C5- and unselected cells. W5C5+ cells differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and endothelial cells. W5C5+ cells produce endometrial stromal-like tissue in vivo. In terms of clonogenicity, magnetic bead-selected W5C5+ cells gave rise to significantly higher CFU numbers compared to flow-sorted W5C5+ cells. This study identified W5C5 as a single marker capable of purifying eMSCs possessing MSC properties and reconstituting endometrial stromal tissues in vivo. W5C5 enriches eMSCs to high purity and provides a simple protocol for their prospective isolation using magnetic bead selection rather than flow sorting. W5C5 selection may provide an alternate, readily available autologous source of MSC, obtainable with minimal morbidity using an office endometrial biopsy procedure for future cell-based therapies.
Detrimental graft-versus-host disease (GVHD) still remains a major cause of death in hematopoietic stem cell transplantation (HSCT). The recently explored depletion of naive cells from mobilized ...grafts (CD45RA depletion) has shown considerable promise, yet is unable to eliminate the incidence of GVHD. Analysis of CD45RA-depleted haploidentical mixed lymphocytes culture (haplo-MLC) revealed insufficient suppression of alloresponses in the CD4
compartment and identified CD276 as a marker for alloreactive memory Th1 T cells. Conclusively, depleting CD276
cells from CD45RA-depleted haplo-MLC significantly attenuated alloreactivity to recipient cells while increasing antiviral reactivity and maintaining anti-third party reactivity in vitro. To evaluate these findings in vivo, bulk, CD45RA-depleted, or CD45RA/CD276-depleted CD4
T cells from HLA-DR4
healthy humans were transplanted into NSG-Ab°DR4 mice, a sensitive human allo-GVHD model. Compellingly, CD45RA/CD276-depleted grafts from HLA-DR4
donors or in vivo depletion of CD276
cells after transplant of HLA-DR4
memory CD4 T cells significantly delay the onset of GVHD symptoms and significantly alleviate its severity in NSG-Ab°DR4 mice. The clinical courses correlated with diminished Th1-cytokine secretion and downregulated CXCR6 expression of engrafted peripheral T cells. Collectively, mismatched HLA-mediated GVHD can be controlled by depleting recipient-specific CD276
alloreacting T cells from the graft, highlighting its application in haplo-HSCT.
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.
1 University Clinic of Tübingen, Department of Internal Medicine II, Division of Hematology, Oncology, and Immunology, Tübingen
2 University of Tübingen, Institute of Anatomy, Department of ...Experimental Embryology, Division of Tissue Engineering, Tübingen
3 University Clinic of Tübingen, Childrens Hospital, Department of General Pediatrics, Division of Hematology and Oncology, Tübingen
4 Leiden University Medical Center, Department of Immunohematology and Blood Transfusion, Center for Stem Cell Therapy, Leiden, The Netherlands and
5 Hospital for Workers Compensation Tübingen, Department of Orthopedic Surgery, Tübingen, Germany
Correspondence: Hans-Jörg Bühring, Ph.D., University of Tübingen, Department of Internal Medicine II, Medical, Otfried-Müller-Str. 10, 72076, Tübingen, Germany., E-mail: hans-joerg.buehring{at}uni-tuebingen.de
Background: Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells.
Design and Methods: Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining.
Results: Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90-fold enriched in the MSCA-1 + CD56 – fraction and ~180-fold in the MSCA-1 + CD56 + fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1 + CD56 – mesenchymal stem cells subset and CD166 to MSCA-1 + CD56 ± mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1 + CD56 ± cells whereas adipocytes emerged exclusively from MSCA-1 + CD56 – cells. The culture of single sorted MSCA-1 + CD56 + cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities.
Conclusions: Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1 + CD56 + subset is an attractive starting population for autologous chondrocyte transplantation.
Key words: mesenchymal stem cells, CD56, MSCA-1.
Prospective isolation of human MSC Harichandan, Abhishek; Bühring, Hans-Jörg, PhD
Best practice & research. Clinical haematology,
03/2011, Letnik:
24, Številka:
1
Journal Article
Recenzirano
Conventionally, mesenchymal/stromal stem cells (MSC) are functionally isolated from primary tissue based on their capacity to adhere to the plastic surface. This isolation procedure is hampered by ...the unpredictable influence of secreted molecules or interactions with co-cultured hematopoietic and other unrelated cells as well as by the arbitrarily selected removal time of non-adherent cells prior to expansion of MSC. Early removal of non-adherent cells may result in the elimination of a late adhering MSC subsets and late removal increases the influence of undesired cells on the growth and differentiation of MSC. Finally, in conventional protocols MSC are co-expanded together with macrophages, endothelial cells and other adherent cells. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSC based on the selective expression or absence of surface markers. Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow-derived MSC.
Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid ...morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III β-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.
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