Sabrina Treml

Novel Markers for the Prospective Isolation of Human MSC

Abstract:  The isolation of mesenchymal stem cells (MSC) from primary tissue is hampered by the limited selectivity of available markers. So far, CD271 is one of the most specific markers for bone marrow (BM)‐derived MSC. In search of additional markers, monoclonal antibodies (mAbs) with specificity for immature cells were screened by flow cytometry for their specific reactivity with the rare CD271+ population. The recognized CD271+ populations were fractionated by fluorescence‐activated cell sorting and the clonogenic capacity of the sorted cells was analyzed for their ability to give rise to CFU‐F. The results showed that only the CD271bright but not the CD271dim population contained CFU‐F. Two‐color flow cytometry analysis revealed that only the CD271bright population was positive for the established MSC markers CD10, CD13, CD73, and CD105. In addition, a variety of mAbs specific for novel and partially unknown antigens selectively recognized the CD271bright population but no other BM cells. The new MSC‐specific molecules included the platelet‐derived growth factor receptor‐β (CD140b), HER‐2/erbB2 (CD340), frizzled‐9 (CD349), the recently described W8B2 antigen, as well as cell‐surface antigens defined by the antibodies W1C3, W3D5, W4A5, W5C4, W5C5, W7C6, 9A3, 58B1, F9‐3C2F1, and HEK‐3D6. In conclusion, the described markers are suitable for the prospective isolation of highly purified BM‐MSC. These MSC may be used as an improved starting population for transplantation in diseases like osteogenesis imperfecta, cartilage repair, and myocardial infarction.

Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1

Mesenchymal stem cells are self-renewing cells with the ability to differentiate into osteocytes, chondrocytes and adipocytes. This article describes a subset of mesenchymal stem cells with distinct phenotypic and functional properties. 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.

Phenotypic Characterization of Distinct Human Bone Marrow–Derived MSC Subsets

Very recently, we identified two distinct mesenchymal stem cell (MSC) subsets in primary bone marrow (BM) that differ in their expression pattern (CD271brightMSCA‐1dimCD56+ and CD271brightMSCA‐1brightCD56−) and morphology as well as in their clonogenic and differentiation capacity. Here we analyzed the cell surface antigen expression in these subsets in more detail and compared the profiles with the expression pattern on cultured MSCs. Most of the tested antigens, including CD13, CD15, CD73, CD140b, CD144, CD146, and CD164, are expressed at similar levels in both primary BM populations. However, a number of markers were differentially expressed. Of these, CD166 (ALCAM), CD200, and CD106 (VCAM‐1) showed an almost selective expression on either CD271brightMSCA‐1dimCD56+ (increased CD166 and CD200 expression) or CD271brightMSCA‐1brightCD56− (increased CD106 expression) MSCs, respectively. Additional markers with elevated expression on CD56+ MSCs include F9‐3C2F1, HEK‐3D3, HEK5‐1B3, and W1C3 antigens, whereas CD10, CD26, CD106, 7C5G1, 9A3G2, 56A1C2, 66E2D11, HEK‐3D6, HEK4‐1A1, HEK4‐2D6, W1D6, W4A5, W7C6, and W8B2 (MSCA‐1) antigens showed increased expression in the CD56− population. The majority of the analyzed markers found on primary MSCs were also expressed on cultured MSCs. However, in contrast to primary MSCs, HEK7‐1C4, W1C3, W1D6, and W4A5 antigens were absent on the cultured counterparts. 7G5G1 and 9A3G2 antigens showed reduced, and HEK‐3D6, F9‐3C2, and HEK‐3D3 showed increased expression on cultured cells. The extended knowledge about the phenotype of the two subsets and the identification of novel MSC markers may result in the isolation of attractive starting populations for applications in regenerative medicine.

Evaluation of the osteogenic and chondrogenic differentiation capacities of equine adipose tissue-derived mesenchymal stem cells

OBJECTIVE To evaluate the proliferative behavior, telomere length, immunophenotype, and differentiation capacity of equine adipose tissue-derived mesenchymal stem cells (AT-MSCs). ANIMALS 6 adult racing horses treated for articular Injury but otherwise healthy. PROCEDURES AT-MSCs were Isolated from horses and expanded In Dulbecco modified Eagle medium enriched with fetal bovine serum and antimicrobials. Expression of cell surface antigens and telomere length were Investigated via flow cytometry Differentiation of MSCs Into chondrocytes, osteoblasts, and adipocytes was Induced In vitro by specific stimuli and was evaluated by analyzing marker genes with quantitative reverse transcriptase PCR assays and immunocytochemical and cytologie evaluations. RESULTS Equine MSCs could be cultured up to the fifth passage before signs of senescence, apoptosis, and detachment Indicated cellular exhaustion. However, the AT-MSCs from 2 of 6 horses survived to later passages with Increased doubling rates and telomere lengths. The cells had a typical phenotype, with expression of CD14, CD73, CD90, CD105, CD140b, and CD164 antigens and a lack of CD34 and CD45 antigens. The cells also had a strong potential to differentiate Into osteoblasts, as characterized by Intense von Kossa and alizarin red staining as well as high Induction of osteopontin. Chondrogenic differentiation was detected via Alelan blue staining and expression of aggrecan and type II collagen Adipogenesis was Induced in AT-MSCs by supplementation of differentiation media with rabbit serum. CONCLUSIONS AND CLINICAL RELEVANCE Equine AT-MSCs representa suitable cellular source for regenerative treatment of bone or cartilage defects, particularly when expanded In vitro for only a few passages.

The mesenchymal stem cell antigen MSCA-1 is identical to tissue non-specific alkaline phosphatase.

We have recently identified 2 distinct CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-) MSC subsets in primary femur-derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here, we show that MSCA-1 is identical to tissue non-specific alkaline phosphatase (TNAP), an ectoenzyme known to be expressed at high levels in liver, bone, and kidney as well as in embryonic stem (ES) cells. SDS-PAGE of WERI-RB-1 cell lysate and supernatant from phosphatidylinositol-specific phospholipase C (PI-PLC)-treated WERI-RB-1 cells resulted in the appearance of a prominent 68-kDa band. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) sequence analysis revealed TNAP-specific peptides. Screening of the MSCA-1-specific antibody W8B2 on HEK-293 cells transfected with the full-length coding sequence of TNAP showed specific reactivity with transfected but not with parent cell line. In addition, TNAP-specific mRNA expression was selectively detected in the transfectant line. In agreement with these findings, enzymatic activity of TNAP was exclusively detected in sorted MSCA-1(+) BM cells but not in the MSCA-1(-) negative fraction. Surface marker analysis revealed coexpression of the embryonic marker SSEA-3 but not SSEA-4, TRA-1-60, and TRA-1-81. In endometrium, TNAP is expressed at intermediate levels on CD146(+) cells and at high levels in the luminal space of glandular epithelia. Our results demonstrate that TNAP is a selective marker for the prospective isolation of BM-derived MSC and MSC-like cells in endometrium.