Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells

Zhang,Wei; Hu,Jiabo; Ma,Quanhui; Hu,Sanqiang; Wang,Yanyan; Wen,Xiangmei; Ma,Yongbin; Xu,Hong; Qian,Hui; Xu,Wenrong

doi:10.3892/etm.2014.1801

Cryopreserved mouse fetal liver stromal cells treated with mitomycin C are able to support the growth of human embryonic stem cells

Authors:
- Wei Zhang
- Jiabo Hu
- Quanhui Ma
- Sanqiang Hu
- Yanyan Wang
- Xiangmei Wen
- Yongbin Ma
- Hong Xu
- Hui Qian
- Wenrong Xu
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Affiliations: School of Medical Science and Laboratory Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, P.R. China, Department of Clinical Laboratory, Jiangsu Provincial Hospital on Integration of Chinese and Western Medicine, Nanjing, Jiangsu 210028, P.R. China, Maternal and Child Care Service Centre of Lianyungang, Lianyungang, Jiangsu 222006, P.R. China, Department of Clinical Laboratory, Zhenjiang Centre for Disease Prevention and Control, Zhenjiang, Jiangsu 212003, P.R. China
Published online on: June 23, 2014 https://doi.org/10.3892/etm.2014.1801
Pages: 935-942

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Abstract

An immortalized mouse fetal liver stromal cell line, named KM3, has demonstrated the potential to support the growth and maintenance of human embryonic stem cells (hESCs). In this study, the characteristics of KM3 cells were examined following cryopreservation at -70˚C and in liquid nitrogen for 15, 30 and 60 days following treatment with 10 µg/ml mitomycin C. In addition, whether the KM3 cells were suitable for use as feeder cells to support the growth of hESCs was evaluated. The inhibition of mitosis without cell death was observed when the KM3 cells were treated with 10 µg/ml mitomycin C for 2 h. The morphology of the KM3 cells cryopreserved in liquid nitrogen for 60 days was not markedly changed, and the cell survival rate was 84.60±1.14%. By contrast, the survival rate of the KM3 cells was 66.40±2.88% following cryopreservation at -70˚C for 60 days; the cells readily detached, were maintained for a shorter time, and had a reduced expression level of basic fibroblast growth factor. hESCs cultured on KM3 cells cryopreserved in liquid nitrogen for 60 days showed the typical bird's nest structure, with clear boundaries and a differentiation rate of 16.33±2.08%. The differentiation rate of hESCs cultured on KM3 cells cryopreserved at -70˚C for 60 days was 37.67±3.51%. These results indicate that the cryopreserved KM3 cells treated with mitomycin C may be directly used in the subculture of hESCs, and the effect is relatively good with -70˚C short-term or liquid nitrogen cryopreservation.

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1	Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al: Embryonic stem cell lines derived from human blastocysts. Science. 282:1145–1147. 1998. View Article : Google Scholar : PubMed/NCBI
2	Niwa H: How is pluripotency determined and maintained? Development. 134:635–646. 2007. View Article : Google Scholar : PubMed/NCBI
3	Darabi R and Perlingeiro RC: Lineage-specific reprogramming as a strategy for cell therapy. Cell Cycle. 7:1732–1737. 2008. View Article : Google Scholar : PubMed/NCBI
4	Reubinoff BE, Pera MF, Fong CY, et al: Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 18:399–404. 2000. View Article : Google Scholar : PubMed/NCBI
5	Hovatta O, Mikkola M, Gertow K, et al: A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod. 18:1404–1409. 2003. View Article : Google Scholar : PubMed/NCBI
6	Cheng L, Hammond H, Ye Z, et al: Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells. 21:131–142. 2003. View Article : Google Scholar : PubMed/NCBI
7	Inzunza J, Gertow K, Strömberg MA, et al: Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells. 23:544–549. 2005. View Article : Google Scholar : PubMed/NCBI
8	Lim JW and Bodnar A: Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics. 2:1187–1203. 2002. View Article : Google Scholar : PubMed/NCBI
9	Cai J, Chen J, Liu Y, et al: Assessing self-renewal and differentiation in human embryonic stem cell lines. Stem Cells. 24:516–530. 2006. View Article : Google Scholar : PubMed/NCBI
10	Chin AC, Fong WJ, Goh LT, et al: Identification of proteins from feeder conditioned medium that support human embryonic stem cells. J Biotechnol. 130:320–328. 2007. View Article : Google Scholar : PubMed/NCBI
11	Levenstein ME, Ludwig TE, Xu RH, et al: Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells. 24:568–574. 2006. View Article : Google Scholar : PubMed/NCBI
12	Wang G, Zhang H, Zhao Y, et al: Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers. Biochem Biophys Res Commun. 330:934–942. 2005. View Article : Google Scholar : PubMed/NCBI
13	Park Y, Kim JH, Lee SJ, et al: Human feeder cells can support the undifferentiated growth of human and mouse embryonic stem cells using their own basic fibroblast growth factors. Stem Cells Dev. 20:1901–1910. 2011. View Article : Google Scholar : PubMed/NCBI
14	Lin S and Talbot P: Methods for culturing mouse and human embryonic stem cells. Methods Mol Biol. 690:31–56. 2011. View Article : Google Scholar : PubMed/NCBI
15	Hu J, Hu S, Ma Q, et al: Immortalized mouse fetal liver stromal cells support growth and maintenance of human embryonic stem cells. Oncol Rep. 28:1385–1391. 2012.PubMed/NCBI
16	Talbot MJ and White RG: Cell surface and cell outline imaging in plant tissues using the backscattered electron detector in a variable pressure scanning electron microscope. Plant Methods. 9:402013. View Article : Google Scholar
17	Lee DY, Lee MK, Kim GS, et al: Brazilin inhibits growth and induces apoptosis in human glioblastoma cells. Molecules. 18:2449–2457. 2013. View Article : Google Scholar : PubMed/NCBI
18	Wang N, Ren GD, Zhou Z, et al: Cooperation of myocardin and Smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells. IUBMB Life. 64:331–339. 2012. View Article : Google Scholar : PubMed/NCBI
19	Li C, Yang Y, Lu X, et al: Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos. In Vitro Cell Dev Biol Anim. 46:186–191. 2010. View Article : Google Scholar : PubMed/NCBI
20	Brockbank KG, Carpenter JF and Dawson PE: Effects of storage temperature on viable bioprosthetic heart valves. Cryobiology. 29:537–542. 1992. View Article : Google Scholar : PubMed/NCBI
21	Galmes A, Besalduch J, Bargay J, et al: Long-term storage at −80 degrees C of hematopoietic progenitor cells with 5-percent dimethyl sulfoxide as the sole cryoprotectant. Transfusion. 39:70–73. 1999.
22	Massie I, Selden C, Hodgson H and Fuller B: Storage temperatures for cold-chain delivery in cell therapy: a study of alginate-encapsulated liver cell spheroids stored at −80°C or −170°C for up to 1 year. Tissue Eng Part C Methods. 19:189–195. 2013.PubMed/NCBI
23	Lu J, Hou R, Booth CJ, et al: Defined culture conditions of human embryonic stem cells. Proc Natl Acad Sci USA. 103:5688–5693. 2006. View Article : Google Scholar : PubMed/NCBI
24	Murai T, Sato M, Nishiyama H, et al: Ultrastructural analysis of nanogold-labeled cell surface microvilli in liquid by atmospheric scanning electron microscopy and their relevance in cell adhesion. Int J Mol Sci. 14:20809–20819. 2013. View Article : Google Scholar
25	Ubelmann F, Chamaillard M, El-Marjou F, et al: Enterocyte loss of polarity and gut wound healing rely upon the F-actin-severing function of villin. Proc Natl Acad Sci USA. 110:E1380–E1389. 2013. View Article : Google Scholar : PubMed/NCBI
26	Ozolek JA, Jane EP, Esplen JE, et al: In vitro neural differentiation of human embryonic stem cells using a low-density mouse embryonic fibroblast feeder protocol. Methods Mol Biol. 584:71–95. 2010. View Article : Google Scholar : PubMed/NCBI
27	Heng BC, Liu H and Cao T: Feeder cell density - a key parameter in human embryonic stem cell culture. In Vitro Cell Dev Biol Anim. 40:255–257. 2004. View Article : Google Scholar : PubMed/NCBI
28	Park SP, Lee YJ, Lee KS, et al: Establishment of human embryonic stem cell lines from frozen-thawed blastocysts using STO cell feeder layers. Hum Reprod. 19:676–684. 2004. View Article : Google Scholar : PubMed/NCBI
29	Hou P, Li Y, Zhang X, et al: Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science. 341:651–654. 2013. View Article : Google Scholar : PubMed/NCBI
30	Maherali N and Hochedlinger K: Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell. 3:595–605. 2008. View Article : Google Scholar : PubMed/NCBI