Mesenchymal stem cells, or MSCs, are fibroblastoid multipotent adult stem cells, with a high self-renewal potential (Figure 1). These cells have been derived from numerous human tissues; including adipose tissue, tendons, the umbilical cord matrix, bone marrow, lung tissue, and the periosteum [2, 6]. Researchers are using several techniques for cell isolation and expansion, and are carrying out several different methods for characterizing their MSCs. These discrepancies in defining the characteristics of MSCs complicate any comparisons of results between studies - researchers cannot be sure that cells employed in other studies are sufficiently similar enough.
Figure 1. An overview of mesenchymal stem cell (MSC) development. MSCs can be derived from several sources, but have recently been shown to originate from the perivascular niche . They show specific stem cell characteristics such as self-renewal and multipotency.
As per recent investigations, in the United States alone, billions of dollars are spent every year on preclinical research that cannot be reproduced . Use of different biological reagents, inconsistent reference materials, and poor study design are some of the factors contributing to the lack of reproducibility in preclinical experiments. By using validated research materials and technologies, on top of the best available practices, the reproducibility of basic and preclinical research can be considerably enhanced, thereby accelerating improvement in the field and assisting the development of clinical therapies.
To deal with these problems, the International Society for Cellular Therapy (ISCT) has set three minimum criteria (Figure 2) to guarantee the integrity and clear identification of human MSCs and to offer a common set of comparable standard criteria for MSC research .
Figure 2. Summary of the ISCT criteria for identifying MSCs for research purposes. (1) MSCs must be plastic-adherent under standard culture conditions. (2) MSCs must express the surface antigens CD105, CD73, and CD90. A lack of expression of hematopoietic antigens (CD45, CD34, CD14/CD11b, CD79a/CD19, HLA-DR) is recommended, along with a minimum purity of ≥95% for CD105, CD73, and CD90 positive cells and ≤2% expression of hematopoietic antigens. (3) MSCs must be shown to be multipotent and be able to give rise to adipocytes, osteoblasts, and chondrocytes under the standard in vitro tissue culture-differentiating conditions.
As per these instructions, MSCs have to be plastic-adherent when maintained under standard culture conditions and express precise surface markers (Table 1). Moreover, under the standard in vitro differentiating conditions, MSCs must be able to differentiate into adipocytes, chondrocytes, and osteoblasts (Figure 2).
Table 1. List of cell surface antigens recommended by the ISCT for identifying MSCs
||Originally recognized by MAb SH2; endothelial cells, MSCs, hematopoietic cells
||ecto 5‘ nucleotidase
||Originally recognized by MAb SH3 and SH4; B- and T-cell subsets, dendritic reticulum cells, epithelial and endothelial cells
||Hematopoietic cells, neuronal cells, fibroblasts, stromal cells, activated endothelial cells
||Primitive hematopoietic progenitors and endothelial cells
|CD14 / CD11b
||– / integrin α-M, CR3A
||Monocytes, macrophages, Langerhans cells and granulocytes/granulocytes, monocytes, natural killer cells, T- and B-cells and dendritic cells
|CD79α / CD19
||MB1, IGA / –
||B-cells / B-cells and follicular dendritic cells
||Not expressed on MSCs, unless stimulated, e.g., by IFN-γ
Sources: ISCT (International Society for Cellular Therapy), Abcam Human CD Antigen Guide.
When MSC surface markers were characterized by flow cytometry (with subsequent isolation from bone marrow) using PromoCell’s MSC Growth Medium, they demonstrated a defined MSC population based on the suggested ISCT markers for determining MSC identity (Figure 3A and Figure 3B illustrate the positive and negative markers, respectively)
Figure 3. Flow cytometry analysis of PromoCell human primary mesenchymal cells isolated from bone marrow. (A) Histograms of CD73, CD90, and CD105 expression. MSCs show a defined population that is positive for the mentioned markers. (B) Histograms of CD14, CD19, CD45, and HLA-DR exemplarily represent a defined population that is negative for the mentioned endothelial and hematopoietic markers. The results conform to the ISCT guidelines (see Figure 2 and Table 1)
Using the PromoCell MSC Growth Medium DXF, the expansion of bone marrow-derived MSCs yielded a stable growth performance over different passages (Figure 4). The ISCT criteria (see Figure 2) are fulfilled by all of the MSCs used in this research, thus showing the self-renewal potentials of these defined cell populations.
Figure 4. Growth performance of hMSCs isolated from bone marrow (hMSC-BM) on fibronectin-coated tissue culture plastic. (A) The MSCs were cultured with PromoCell Growth Medium DXF, which has a defined and xeno-free formula (MSC-GM DXF). (B) The cumulative numbers of population doublings and doubling times are shown here over seven passages. A stable growth rate of less than 30 hours/doubling can be observed even after prolonged in vitro culture for 32 population doublings over the course of seven passages.
Using PromoCell MSC differentiation media, differentiation of expanded bone marrow MSCs into chondrocytes, adipocytes, and osteoblasts, according to the ISCT criteria, was assayed in passage 3 (Figure 5). All of the MSCs that were tested differentiated effectively into the three types of cells, thus exhibiting their multipotency. Adipogenic differentiation shows the wide formation of intracellular lipid vacuole, unique to mature adipocytes (Figure 5A). Cartilage spheroid formation was induced to determine chondrogenic isolation of MSCs (Figure 5B). Lastly, the differentiation of MSCs into mature osteoblasts was shown by Alizarin Red S staining of extracellular calcium deposits in mineralized cells (Figure 5C).
Figure 5. Differentiation of in vitro cultured PromoCell human MSCs into adipocytes (A), chondrocytes (B), and osteoblasts (C). (A) Lipid vesicle accumulation in adipocytes differentiated of human MSCs derived from bone marrow. The culture exhibits extensive intracellular lipid vacuole formation typical for mature adipocytes. (B) Alcian Blue staining of MSC spheroids after in vitro differentiation into cartilage. Induced spheroids exhibit an intensely blue color indicative for cartilage extracellular matrix. (C) Alizarin Red S staining of extracellular calcium deposits in mineralized hMSC-BM-derived mature osteoblasts.
Conclusions and Future Prospects
Isolating human MSCs using PromoCell Media offers a well-defined, uniform MSC population consistent with the marker expression profiles defined by the ISCT. The stable growth performance and expansion demonstrate the self-renewing potentials of these cells over the course of several passages. The differentiation of expanded MSCs has demonstrated multipotency into chondrogenic, adipogenic, and osteogenic lineages. On the whole, this data signifies that validation of cellular identity based on ISCT recommendations results in a well-defined MSC population, ideal for in vitro expansion while retaining stemness.
The MSC isolation technique and growth medium used for culturing have a vital role to play in obtaining a clear MSC population that agrees with the definition provided in the ISCT principles.
Standardization of the characterization and culturing of MSCs offers valuable scientific data, as well as assuring comparability across several laboratories. It could promote quicker growth in preclinical studies as well as faster translation into the continuous development of clinical applications .
Furthermore, the use of MSCs from certified sources is becoming a growing demand, as this often has to be proved to obtain financial support for preclinical research projects. For instance, the National Institutes of Health (NIH) in the United States recently made its approval process and review criteria more stringent to make sure that only the most accurate, transparent, and robust research is funded; enhancing the reproducibility of discoveries .
- Freedman LP, Cockburn IM, Simcoe TS. The economics of reproducibility in preclinical research. PLoS Biol. 2015 13(6):e1002165. doi: 10.1371/journal.pbio.1002165.
- da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells. 2008 26(9):2287–99.
- Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008 3(3):301–13.
- Dominici M, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytother. 2006 8(4):315–7.
- National Institutes of Health (NIH), Notice Number: NOT-OD-15-103.
- Ullah, I, Subbarao RB and Rho GJ. Human mesenchymal stem cells - current trends and future prospective. Bioscience Reports. 2015 35, e00191, BSR20150025.
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