Cell and Tissue Research

, Volume 366, Issue 1, pp 113–127 | Cite as

Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates

  • Chizuka ObaraEmail author
  • Ken-ichi Tomiyama
  • Kazuya Takizawa
  • Rafiqul Islam
  • Takeshi Yasuda
  • Takaya Gotoh
  • Katsushi TajimaEmail author
Regular Article


Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.


Nanoculture plate Mouse bone marrow mesenchymal stem/stromal cells 3-D aggregate culture Alpha-fetoprotein Tissue engineering 



We thank Ms. S. Fukuzaki, N. Gotoh, K. Noshiro, and Mr. M. Hama for expert technical assistance, and Drs. I. Tanaka, H. Ishihara, H. Yakumaru, M. Hazawa, and Y. Michikawa for helpful suggestions. We are also grateful to the FACS support team of the National Institute of Radiological Sciences for their technical support regarding the flow cytometry experiments.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

441_2016_2405_MOESM1_ESM.pdf (2.7 mb)
ESM 1 (PDF 2,735 kb)


  1. Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R (2000) Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 28:707–715CrossRefPubMedGoogle Scholar
  2. Baraniak PR, McDevitt TC (2012) Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential. Cell Tissue Res 347:701–711CrossRefPubMedGoogle Scholar
  3. Bartosh TJ, Ylostalo JH, Mohammadipoor A, Bazhanov N, Coble K, Claypool K, Lee RH, Choi H, Prockop DJ (2010) Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107:13724–13729CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B (2006) Aging of mesenchymal stem cell in vitro. BMC Cell Biol 7:14CrossRefPubMedPubMedCentralGoogle Scholar
  5. Burdon TJ, Paul A, Noiseux N, Prakash S, Shum-Tim D (2011) Bone marrow stem cell derived paracrine factors for regenerative medicine: current perspectives and therapeutic potential. Bone Marrow Res 2011:207326CrossRefPubMedGoogle Scholar
  6. Caplan AI (2007) Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol 213:341–347CrossRefPubMedGoogle Scholar
  7. Cheng NC, Wang S, Young TH (2012) The influence of spheroid formation of human adipose-derived stem cells on chitosan films on stemness and differentiation capabilities. Biomaterials 33:1748–1758CrossRefPubMedGoogle Scholar
  8. Cheng NC, Chen SY, Li JR, Young TH (2013) Short-term spheroid formation enhances the regenerative capacity of adipose-derived stem cells by promoting stemness, angiogenesis, and chemotaxis. Stem Cells Transl Med 2:584–594CrossRefPubMedPubMedCentralGoogle Scholar
  9. Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S (2011) Role of YAP/TAZ in mechanotransduction. Nature 474:179–183CrossRefPubMedGoogle Scholar
  10. Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126:677–689CrossRefPubMedGoogle Scholar
  11. Folkman J, Hochberg M (1973) Self-regulation of growth in three dimensions. J Exp Med 138:745–753CrossRefPubMedPubMedCentralGoogle Scholar
  12. Ge J, Guo L, Wang S, Zhang Y, Cai T, Zhao RC, Wu Y (2014) The size of mesenchymal stem cells is a significant cause of vascular obstructions and stroke. Stem Cell Rev 10:295–303CrossRefPubMedGoogle Scholar
  13. Ghaedi M, Soleimani M, Shabani I, Duan Y, Lotfi AS (2012) Hepatic differentiation from human mesenchymal stem cells on a novel nanofiber scaffold. Cell Mol Biol Lett 17:89–106CrossRefPubMedGoogle Scholar
  14. Hamazaki T, Oka M, Yamanaka S, Terada N (2004) Aggregation of embryonic stem cells induces Nanog repression and primitive endoderm differentiation. J Cell Sci 117:5681–5686CrossRefPubMedGoogle Scholar
  15. Kanda Y (2013) Investigation of the freely available easy-to-use software “EZR” for medical statistics. Bone Marrow Transplant 48:452–458CrossRefPubMedGoogle Scholar
  16. Kazemnejad S, Allameh A, Soleimani M, Gharehbaghian A, Mohammadi Y, Amirizadeh N, Jazayery M (2009) Biochemical and molecular characterization of hepatocyte-like cells derived from human bone marrow mesenchymal stem cells on a novel three-dimensional biocompatible nanofibrous scaffold. J Gastroenterol Hepatol 24:278–287CrossRefPubMedGoogle Scholar
  17. Knight E, Przyborski S (2014) Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J Anat 227:746–756CrossRefPubMedGoogle Scholar
  18. Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, Chen JR, Chen YP, Lee OK (2004) In vitro hepatic differentiation of human mesenchymal stem cells. Hepatology 40:1275–1284CrossRefPubMedGoogle Scholar
  19. Li Y, Guo G, Li L, Chen F, Bao J, Shi YJ, Bu H (2015) Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance. Cell Tissue Res 360:297–307CrossRefPubMedGoogle Scholar
  20. Makino S, Fukuda K, Miyoshi S, Konishi F, Kodama H, Pan J, Sano M, Takahashi T, Hori S, Abe H, Hata J, Umezawa A, Ogawa S (1999) Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103:697–705CrossRefPubMedPubMedCentralGoogle Scholar
  21. Miyagawa Y, Okita H, Hiroyama M, Sakamoto R, Kobayashi M, Nakajima H, Katagiri YU, Fujimoto J, Hata J, Umezawa A, Kiyokawa N (2011) A microfabricated scaffold induces the spheroid formation of human bone marrow-derived mesenchymal progenitor cells and promotes efficient adipogenic differentiation. Tissue Eng Part A 17:513–521CrossRefPubMedGoogle Scholar
  22. Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E, Suzuki S, Miyauchi-Hara C, Nagoshi N, Sunabori T, Shimmura S, Miyawaki A, Nakagawa T, Suda T, Okano H, Matsuzaki Y (2009) Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med 206:2483–2496CrossRefPubMedPubMedCentralGoogle Scholar
  23. Nakamura K, Kato N, Aizawa K, Mizutani R, Yamauchi J, Tanoue A (2011) Expression of albumin and cytochrome P450 enzymes in HepG2 cells cultured with a nanotechnology-based culture plate with microfabricated scaffold. J Toxicol Sci 36:625–633CrossRefPubMedGoogle Scholar
  24. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147CrossRefPubMedGoogle Scholar
  25. Prockop DJ (1997) Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276:71–74CrossRefPubMedGoogle Scholar
  26. Rettinger CL, Fourcaudot AB, Hong SJ, Mustoe TA, Hale RG, Leung KP (2014) In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates. Cell Tissue Res 358:395–405CrossRefPubMedGoogle Scholar
  27. Sart S, Tsai AC, Li Y, Ma T (2014) Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications. Tissue Eng Part B Rev 20:365–380CrossRefPubMedGoogle Scholar
  28. Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith AG, Nishikawa S (2007) Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129:1377–1388CrossRefPubMedGoogle Scholar
  29. Tuan RS, Boland G, Tuli R (2003) Adult mesenchymal stem cells and cell-based tissue engineering. Arthritis Res Ther 5:32–45CrossRefPubMedGoogle Scholar
  30. Wagner W, Bork S, Lepperdinger G, Joussen S, Ma N, Strunk D, Koch C (2010) How to track cellular aging of mesenchymal stromal cells? Aging 4:224–230CrossRefGoogle Scholar
  31. Wei X, Yang X, Han ZP, Qu FF, Shao L, Shi YF (2013) Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol Sin 34:747–754CrossRefPubMedPubMedCentralGoogle Scholar
  32. Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61:364–370CrossRefPubMedGoogle Scholar
  33. Yamada KM, Cukierman E (2007) Modeling tissue morphogenesis and cancer in 3D. Cell 130:601–610CrossRefPubMedGoogle Scholar
  34. Yamaguchi Y, Ohno J, Sato A, Kido H, Fukushima T (2014) Mesenchymal stem cell spheroids exhibit enhanced in-vitro and in-vivo osteoregenerative potential. BMC Biotechnol 14:105CrossRefPubMedPubMedCentralGoogle Scholar
  35. Yamazoe T, Shiraki N, Toyoda M, Kiyokawa N, Okita H, Miyagawa Y, Akutsu H, Umezawa A, Sasaki Y, Kume K, Kume S (2013) A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells. J Cell Sci 126:5391–5399CrossRefPubMedGoogle Scholar
  36. Zhang Q, Nguyen AL, Shi S, Hill C, Wilder-Smith P, Krasieva TB, Le AD (2012) Three-dimensional spheroid culture of human gingiva-derived mesenchymal stem cells enhances mitigation of chemotherapy-induced oral mucositis. Stem Cells Dev 21:937–947CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Chizuka Obara
    • 1
    Email author
  • Ken-ichi Tomiyama
    • 1
  • Kazuya Takizawa
    • 1
  • Rafiqul Islam
    • 1
  • Takeshi Yasuda
    • 1
  • Takaya Gotoh
    • 1
  • Katsushi Tajima
    • 1
    Email author
  1. 1.Research Center for Radiation Emergency MedicineNational Institute of Radiological SciencesChiba-cityJapan

Personalised recommendations