ISSN 00124966, Doklady Biological Sciences, 2013, Vol. 453, pp. 394–396. © Pleiades Publishing, Ltd., 2013. Original Russian Text © O.V. Payushina, N.N. Butorina, O.N. Sheveleva, S.S. Bukhinnik, E.I. Domaratskaya, 2013, published in Doklady Akademii Nauk, 2013, Vol. 453, No. 5, pp. 574–576.

CELL BIOLOGY

Clonal Growth, Phenotype, and Differentiation Potential of Mesenchymal Stromal Cells Derived from the Rat Fetal Bone O. V. Payushina, N. N. Butorina, O. N. Sheveleva, S. S. Bukhinnik, and E. I. Domaratskaya Presented by Academician M.V. Ugryumov March 28, 2013 Received April 1, 2013

DOI: 10.1134/S0012496613060148

Studies on mesenchymal stromal cells (MSCs) are a focus of modern biology. Initially, MSCs were iso lated from the bone marrow, spleen, and thymus in the form of colonyforming units of fibroblasts (CFUF) [1]; later, MSCs were found in all organs of transient and definitive hematopoiesis. Correlation between the contents of CFUF and hematopoietic stem cells in these organs at various stages of ontogeny may show indirectly consecutive change of location of MSCs, which prepare a niche for hematopoietic cells [2]. It is possible that, during ontogeny, MSCs are modified to some extent. Specifically, in the prenatal period, stro mal cells of the human bone marrow have a higher proliferative activity and differentiation potential compared to those of adult donors [3, 4]; however, they have a lower capability for maintaining hemato poiesis [5]. The problem of changes in MSC properties in ontogeny has been poorly studied. Studies on the features of these cells in hematopoietic organs of developing or mature individuals will shed light on maturation of the hematopoietic system. It is of spe cific interest to describe MSCs, located in the fetal bone, prior to the start of active hematopoiesis and to compare them with stromal cells of the mature bone marrow. This was the purpose of our study. Histological study was performed in sections of the femoral bone of 20day fetuses of Wistar rats. We did not observe clear bone marrow cavity in the femoral bone because the epiphyses consisted of cartilage tis sue, and the diaphyses were filled with nets of bone trabecules, between which, fragments of disintegrated cartilage, multiple mesenchymal cells, and a few hematopoietic cells were located. Cells separated from the bone after its treatment with 0.1% collagenase and seeded into the αMEM medium containing 10% fetal calf serum at a density of (1–2) × 105 cells/mL,

formed a confluent fibroblast monolayer after 3day growing. Discrete colonies formed after seeding at a density of (1–4) × 103 cells/mL and 6 to 12day grow ing. The efficiency of CFUF cloning was more than two orders of value higher compared to cells from the bone marrow of adult rats. Moreover, the activity of alkaline phosphatase, which is specific for osteogenic cells and present in most colonies formed by cells of mature bone marrow, was only rarely observed in the culture of fetal bone CFUF (table). The differences in cloning efficiency of stromal cells obtained from two sources are related to the fact that in contrast to the mature bone marrow mostly consisting of hemato poietic cells, the fetal bone contains mostly mechano cytes, which are capable to clonogenic growth. In addition to the marrow anlage, the periosteum, bone, and cartilage tissues and probably, additions of joint capsule, muscles, and tendons are involved in colony formation by cells of the fetal bone. All these tissues are known as sources of MSCs [6–9], and the number of MSCs in the dense tissues is substantially higher compared to the bone marrow [8, 9].

Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia

Data are presented as means and standard errors for each of three experimental series.

We performed PCR analysis of the contents of CD73, CD90, and CD105 mRNAs, markers of Cloning efficiency of CFUF and the activity of alkaline phosphatase in colonies formed by these cells

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Source of cells Fetal bone

Mature bone marrow

Cloning efficiency, Colonies contain CFUF/1 × 106 cells ing AP+ cells, % 5515.88 ± 1211.51 4708.33 ± 342.13 9819.44 ± 862.75 16.00 ± 1.19 16.77 ± 1.13 13.11 ± 0.89

4.72 ± 1.10 0.59 ± 0.63 7.00 ± 1.08 60.71 ± 3.66 95.71 ± 3.83 94.32 ± 0.58

CLONAL GROWTH, PHENOTYPE, AND DIFFERENTIATION POTENTIAL

(a) GAPDH

CD73

CD90

CD105

(b)

Fig. 1. Expression of MSC markers in primary cultures of fetal bone cells. (a) PCR analysis of CD73, CD90, and CD105 gene expression; (b) staining with monoclonal antibodies to CD90.

human MSCs [10], which were previously found in a culture of stromal cells from the rat bone marrow [11]. We revealed higher expression rates of CD90 and CD105 genes in primary culture of the fetal bone, whereas the content of CD73 mRNA was low or absent (Fig. 1a). Immunocytochemistry also revealed the presence of CD90 (Fig. 1b), but not CD73 in most of cells. Accordingly to some data, CD73 is involved in hematopoiesis [12], and its low expression probably reflects functional immaturity of developing hemato poietic stroma.

(a)

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We estimated the potential of stromal cells from the fetal bone for osteogenesis and adipogenesis in the corresponding induction media [11] and found that these cells differentiated in both directions. After 19– 21 days in culture under the conditions of the osteo genic medium, small dense groups of basophilic cubic cells containing alkaline phosphatase formed. Some of these cell groups exhibited weak cytochemical staining for Ca2+, and in some cells, the specific marker of osteoblasts, osteocalcin, was detected immunocy tochemically. Large mineralized bone nodules, which are typical for osteogenic differentiation of MSCs from the bone marrow of adult rats [11], were practi cally absent. Thus, osteogenic potential of fetal bone cells was relatively low, which is in accordance with the small number of cells containing alkaline phosphatase in colonies formed by this type of CFUF. However, fetal bone cells could be adipogenic in both respective induction medium and osteogenic induction medium and formed adipocytes spontaneously in the absence of any inducers. Single cells or diffuse clusters of cells with inclusions of neutral lipids appeared after 4–7 days in culture; after 10–12 days in culture, the loci of dif ferentiation looked like large groups of round cells with multiple lipid vacuoles (Fig. 2b). The adipogenic potential of MSCs from the fetal bone was similar to those from the mature bone marrow in time of forma tion of adipose cells, their number, and rate of matu rity [11]. Our data show that the phenotypes and potentials of stromal cells that are present in bone tissue at the late stages of prenatal ontogeny correspond to those of MSCs but differ from those of stromal cells of the bone marrow in the low expression of CD73 and low capa bility for osteogenesis. This last characteristic is unex pected because of active formation of bone tissue in fetus extremities. In other studies, MSCs from the fetal bone marrow had higher osteogenic potentials compared to cells from mature donors; however, most

(b)

Fig. 2. Differentiation of MSCs from the fetal bone in the induction media. (a) Osteogenesis, 19 days after induction, cytochem ical staining for AP, nuclei are counterstained with hematoxylin; (b) adipogenesis, 12 days after induction, staining with oil red O and hematoxylin. DOKLADY BIOLOGICAL SCIENCES

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of these studies have been performed on human cells [3, 4]. In addition to the possible speciesspecific dif ferences, inconsistencies in the data may be related to impossibility of separation of pure developing bone marrow from rat fetus without any additions of sur rounding tissues such as bone, cartilage, periosteum, and others. MSCs from these tissues were mostly stud ied during postnatal ontogeny [6–8] and their proper ties, specifically, osteogenic potential, in fetus remained to be less studied. In order to interpret our data, we have to take into account the contribution of cells from these sources. We are going to continue studies on specific features of phenotypes and poten cies of MSCs from the bone marrow and other tissues at various stages of pre and postnatal ontogeny. REFERENCES 1. Friedenstein, A.J., Gorskaja, J.F., and Kulagina, N.N., Exp. Hematol., 1976, vol. 4, no. 5, pp. 267–274. 2. Van Den, Heuvel, R.L., Versele, S.R.M., Schoe ters, G.E.R., and Vanderborght, O.L., Br. J. Haema tol., 1987, vol. 66, no. 1, pp. 15–20.

3. Hu, Y., Ma, L., Jiang, X., and Zhao, C., Zhonghua Xue Ye Xue Za Zhi, 2002, vol. 23, no. 12, pp. 645–648. 4. Guillot, P.V., De Bari, C., Dell’Accio, F., et al., Differ entiation, 2008, vol. 76, no. 9, pp. 946–957. 5. Liu, M., Yang, S.G., Xing, W., et al., Zhonghua Xue Ye Xue Za Zhi, 2011, vol. 19, no. 4, p. 1028. 6. Noth, U., Osyczka, A.M., Tuli, R., et al., J. Orthop. Res., 2002, vol. 20, no. 5, pp. 1060–1069. 7. MirmalekSani, S.H., Tare, R.S., Morgan, S.M., et al., Stem Cells, 2006, vol. 24, no. 4, pp. 1042–1053. 8. Yoshimura, H., Muneta, T., Nimura, A., et al., Cell Tis sue Res., 2007, vol. 327, no. 3, pp. 449–462. 9. Tan, Q., Liu, P.P., Rui, Y.F., and Wong, Y.M., Tissue Eng. Pt. A, 2012, vol. 18, nos. 7–8, pp. 840–851. 10. Dominici, M., Le Blanc, K., Mueller, I., et al., Cyto therapy, 2006, vol. 8, no. 4, pp. 315–317. 11. Kozhevnikova, M.N., Mikaelyan, A.S., and Staros tin, V.I., Tsitologiya, 2009, vol. 51, no. 6, pp. 526–538. 12. Barry, F., Boynton, R., Murphy, M., et al., Biochem. Biophys. Res. Commun., 2001, vol. 289, no. 2, pp. 519– 524.

Translated by M. Stepanichev

DOKLADY BIOLOGICAL SCIENCES

Vol. 453

2013

Clonal growth, phenotype, and differentiation potential of mesenchymal stromal cells derived from the rat fetal bone.

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