Skip to main content

Advertisement

SpringerLink
Log in

A modified preplate technique for efficient isolation and proliferation of mice muscle-derived stem cells

  • Original Article
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

We modified an existing protocol to develop a more efficient method to acquire and culture muscle-derived stem cells (MDSCs) and compared the characteristics of cells obtained from the two methods. This method is based on currently used multistep enzymatic digestion and preplate technique. During the replating process, we replaced the traditional medium with isolation medium to promote fibroblast-like cell adherence at initial replating step, which shortened the purifying duration by up to 4 days. Moreover, we modified the culture container to provide a stable microenvironment that promotes MDSC adherence. We compared the cell morphology, growth curve and the expression of specific markers (Sca-1, CD34, PAX7 and Desmin) between the two cell groups separately obtained from the two methods. Afterwards, we compared the neural differentiation capacity of MDSCs with other muscle-derived cell lineages. The protocol developed here is a fast and effective method to harvest and purify MDSCs from mice limb skeletal muscle.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Agley CC, Rowlerson AM, Velloso CP, Lazarus NL, Harridge SD (2015) Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle. J Vis Exp 95:52049

    Google Scholar 

  • Arsic N, Mamaeva D, Lamb NJ, Fernandez A (2008) Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages. Exp Cell Res 314:1266–1280

    Article  CAS  Google Scholar 

  • Asakura A, Komaki M, Rudnicki M (2001) Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation 68:245–253

    Article  CAS  Google Scholar 

  • Beane OS, Fonseca VC, Cooper LL, Koren G, Darling EM (2014) Impact of aging on the regenerative properties of bone marrow-, muscle-, and adipose-derived mesenchymal stem/stromal cells. PLoS ONE 9:e115963

    Article  Google Scholar 

  • Che X, Guo J, Wang B, Bai Y (2011) Rapid isolation of muscle-derived stem cells by discontinuous Percoll density gradient centrifugation. In Vitro Cell Dev Biol Anim 47:454–458

    Article  CAS  Google Scholar 

  • Chen S, Lewallen M, Xie T (2013) Adhesion in the stem cell niche: biological roles and regulation. Development 140:255–265

    Article  CAS  Google Scholar 

  • Chen B, Ding J, Zhang W, Zhou G, Cao Y, Liu W, Wang B (2016) Tissue engineering of tendons: a comparison of muscle-derived cells, tenocytes, and dermal fibroblasts as cell sources. Plast Reconstr Surg 137:536e–544e

    Article  CAS  Google Scholar 

  • Collins CA, Olsen I, Zammit PS, Heslop L, Petrie A, Partridge TA, Morgan JE (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122:289–301

    Article  CAS  Google Scholar 

  • Cottle BJ, Lewis FC, Shone V, Ellison-Hughes GM (2017) Skeletal muscle-derived interstitial progenitor cells (PICs) display stem cell properties, being clonogenic, self-renewing, and multi-potent in vitro and in vivo. Stem Cell Res Ther 8:158

    Article  Google Scholar 

  • Gharaibeh B, Lu A, Tebbets J, Zheng B, Feduska J, Crisan M, Peault B, Cummins J, Huard J (2008) Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc 3:1501–1509

    Article  CAS  Google Scholar 

  • Gussoni E, Soneoka Y, Strickland CD, Buzney EA, Khan MK, Flint AF, Kunkel LM, Mulligan RC (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401:390–394

    CAS  PubMed  Google Scholar 

  • Han D, Chen S, Fang S, Liu S, Jin M, Guo Z, Yuan Y, Wang Y, Liu C, Mei X (2017) The neuroprotective effects of muscle-derived stem cells via brain-derived neurotrophic factor in spinal cord injury model. Biomed Res Int 2017:1972608

    PubMed  PubMed Central  Google Scholar 

  • Jackson KA, Mi T, Goodell MA (1999) Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci USA 96:14482–14486

    Article  CAS  Google Scholar 

  • Jackson WM, Alexander PG, Bulken-Hoover JD, Vogler JA, Ji Y, McKay P, Nesti LJ, Tuan RS (2013) Mesenchymal progenitor cells derived from traumatized muscle enhance neurite growth. J Tissue Eng Regen Med 7:443–451

    Article  CAS  Google Scholar 

  • Jankowski RJ, Deasy BM, Huard J (2002) Muscle-derived stem cells. Gene Ther 9:642–647

    Article  CAS  Google Scholar 

  • Kallestad KM, McLoon LK (2010) Defining the heterogeneity of skeletal muscle-derived side and main population cells isolated immediately ex vivo. J Cell Physiol 222:676–684

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kalvelyte A, Krestnikova N, Stulpinas A, Bukelskiene V, Bironaite D, Baltriukiene D, Imbrasaite A (2013) Long-term muscle-derived cell culture: multipotency and susceptibility to cell death stimuli. Cell Biol Int 37:292–304

    Article  CAS  Google Scholar 

  • Kelc R, Trapecar M, Vogrin M, Cencic A (2013) Skeletal muscle-derived cell cultures as potent models in regenerative medicine research. Muscle Nerve 47:477–482

    Article  Google Scholar 

  • Khosravi-Farsani S, Amidi F, Habibi Roudkenar M, Sobhani A (2015) Isolation and enrichment of mouse female germ line stem cells. Cell J 16:406–415

    PubMed  PubMed Central  Google Scholar 

  • Lau AM, Tseng YH, Schulz TJ (2014) Adipogenic fate commitment of muscle-derived progenitor cells: isolation, culture, and differentiation. Methods Mol Biol 1213:229–243

    Article  Google Scholar 

  • Lavasani M, Lu A, Thompson SD, Robbins PD, Huard J, Niedernhofer LJ (2013) Isolation of muscle-derived stem/progenitor cells based on adhesion characteristics to collagen-coated surfaces. Methods Mol Biol 976:53–65

    Article  CAS  Google Scholar 

  • Lavasani M, Thompson SD, Pollett JB, Usas A, Lu A, Stolz DB, Clark KA, Sun B, Peault B, Huard J (2014) Human muscle-derived stem/progenitor cells promote functional murine peripheral nerve regeneration. J Clin Invest 124:1745–1756

    Article  CAS  Google Scholar 

  • Lee JY, Qu-Petersen Z, Cao B, Kimura S, Jankowski R, Cummins J, Usas A, Gates C, Robbins P, Wernig A, Huard J (2000) Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol 150:1085–1100

    Article  CAS  Google Scholar 

  • Lee JY, Piao S, Kim IG, Byun SS, Hwang JH, Hong SH, Kim SW, Hwang TK, Lee JY (2012) Effect of human muscle-derived stem cells on cryoinjured mouse bladder contractility. Urology 80:224.e7–224.e11

    Google Scholar 

  • Li Y, Pan H, Huard J (2010) Isolating stem cells from soft musculoskeletal tissues. J Vis Exp. https://doi.org/10.3791/2011

  • Li H, Usas A, Poddar M, Chen CW, Thompson S, Ahani B, Cummins J, Lavasani M, Huard J (2013) Platelet-rich plasma promotes the proliferation of human muscle derived progenitor cells and maintains their stemness. PLoS ONE 8:e64923

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408

    Article  CAS  Google Scholar 

  • Lorant J, Saury C, Schleder C, Robriquet F, Lieubeau B, Negroni E, Leroux I, Chabrand L, Viau S, Babarit C, Ledevin M, Dubreil L, Hamel A, Magot A, Thorin C, Guevel L, Delorme B, Pereon Y, Butler-Browne G, Mouly V, Rouger K (2018) Skeletal muscle regenerative potential of human mustem cells following transplantation into injured mice muscle. Mol Ther 26:618–633

    Article  CAS  Google Scholar 

  • Lu A, Proto JD, Guo L, Tang Y, Lavasani M, Tilstra JS, Niedernhofer LJ, Wang B, Guttridge DC, Robbins PD, Huard J (2012) NF-κB negatively impacts the myogenic potential of muscle-derived stem cells. Mol Ther 20:661–668

    Article  CAS  Google Scholar 

  • Lucas PA, Calcutt AF, Southerland SS, Alan Wilson J, Harvey RL, Warejcka D, Young HE (1995) A population of cells resident within embryonic and newborn rat skeletal muscle is capable of differentiating into multiple mesodermal phenotypes. Wound Repair Regen 3:449–460

    Article  CAS  Google Scholar 

  • Machida S, Spangenburg EE, Booth FW (2004) Primary rat muscle progenitor cells have decreased proliferation and myotube formation during passages. Cell Prolif 37:267–277

    Article  CAS  Google Scholar 

  • Meng J, Chun S, Asfahani R, Lochmuller H, Muntoni F, Morgan J (2014) Human skeletal muscle-derived CD133(+) cells form functional satellite cells after intramuscular transplantation in immunodeficient host mice. Mol Ther 22:1008–1017

    Article  CAS  Google Scholar 

  • Montarras D, Morgan J, Collins C, Relaix F, Zaffran S, Cumano A, Partridge T, Buckingham M (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science 309:2064–2067

    Article  CAS  Google Scholar 

  • Morgan JE, Partridge TA (2003) Muscle satellite cells. Int J Biochem Cell Biol 35:1151–1156

    Article  CAS  Google Scholar 

  • Nakajima N, Tamaki T, Hirata M, Soeda S, Nitta M, Hoshi A, Terachi T (2017) Purified human skeletal muscle-derived stem cells enhance the repair and regeneration in the damaged urethra. Transplantation 101(10):2312–2320

    Article  Google Scholar 

  • Ozasa Y, Gingery A, Thoreson AR, An KN, Zhao C, Amadio PC (2014) A comparative study of the effects of growth and differentiation factor 5 on muscle-derived stem cells and bone marrow stromal cells in an in vitro tendon healing model. J Hand Surg Am 39:1706–1713

    Article  Google Scholar 

  • Park MH, Park JE, Kim MS, Lee KY, Park HJ, Yun JI, Choi JH, Lee E, Lee ST (2014) Development of a high-yield technique to isolate spermatogonial stem cells from porcine testes. J Assist Reprod Genet 31:983–991

    Article  Google Scholar 

  • Qu-Petersen Z, Deasy B, Jankowski R, Ikezawa M, Cummins J, Pruchnic R, Mytinger J, Cao B, Gates C, Wernig A, Huard J (2002) Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 157:851–864

    Article  CAS  Google Scholar 

  • Saito K, Tamaki T, Hirata M, Hashimoto H, Nakazato K, Nakajima N, Kazuno A, Sakai A, Iida M, Okami K (2015) Reconstruction of multiple facial nerve branches using skeletal muscle-derived multipotent stem cell sheet-pellet transplantation. PLoS ONE 10:e0138371

    Article  Google Scholar 

  • Seta H, Maki D, Kazuno A, Yamato I, Nakajima N, Soeda S, Uchiyama Y, Tamaki T (2018) Voluntary exercise positively affects the recovery of long-nerve gap injury following tube-bridging with human skeletal muscle-derived stem cell transplantation. J Clin Med 7:67

    Article  Google Scholar 

  • Spradling A, Drummond-Barbosa D, Kai T (2001) Stem cells find their niche. Nature 414:98–104

    Article  CAS  Google Scholar 

  • Tamaki T, Akatsuka A, Ando K, Nakamura Y, Matsuzawa H, Hotta T, Roy RR, Edgerton VR (2002) Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle. J Cell Biol 157:571–577

    Article  CAS  Google Scholar 

  • Tamaki T, Uchiyama Y, Okada Y, Ishikawa T, Sato M, Akatsuka A, Asahara T (2005) Functional recovery of damaged skeletal muscle through synchronized vasculogenesis, myogenesis, and neurogenesis by muscle-derived stem cells. Circulation 112:2857–2866

    Article  Google Scholar 

  • Tamaki T, Okada Y, Uchiyama Y, Tono K, Masuda M, Nitta M, Hoshi A, Akatsuka A (2008) Skeletal muscle-derived CD34 +/45- and CD34-/45- stem cells are situated hierarchically upstream of Pax7 + cells. Stem Cells Dev 17:653–667

    Article  CAS  Google Scholar 

  • Tamaki T, Tono K, Uchiyama Y, Okada Y, Masuda M, Soeda S, Nitta M, Akatsuka A (2011) Origin and hierarchy of basal lamina-forming and -non-forming myogenic cells in mouse skeletal muscle in relation to adhesive capacity and Pax7 expression in vitro. Cell Tissue Res 344:147–168

    Article  CAS  Google Scholar 

  • Tamaki T, Uchiyama Y, Hirata M, Hashimoto H, Nakajima N, Saito K, Terachi T, Mochida J (2015) Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle–nerve–blood vessel reconstitution. Front Physiol 6:165

    Article  Google Scholar 

  • Tamaki T, Hirata M, Nakajima N, Saito K, Hashimoto H, Soeda S, Uchiyama Y, Watanabe M (2016) A long-gap peripheral nerve injury therapy using human skeletal muscle-derived stem cells (Sk-SCs): an achievement of significant morphological, numerical and functional recovery. PLoS ONE 11:e0166639

    Article  Google Scholar 

  • Vojnits K, Pan H, Dai X, Sun H, Tong Q, Darabi R, Huard J, Li Y (2017) Functional neuronal differentiation of injury-induced muscle-derived stem cell-like cells with therapeutic implications. Sci Rep 7:1177

    Article  Google Scholar 

  • Vukusic K, Jonsson M, Brantsing C, Dellgren G, Jeppsson A, Lindahl A, Asp J (2013) High density sphere culture of adult cardiac cells increases the levels of cardiac and progenitor markers and shows signs of vasculogenesis. Biomed Res Int 2013:696837

    Article  Google Scholar 

  • Wang HD, Guo Q, Quan A, Lopez J, Alonso-Escalante JC, Lough DM, Lee WPA, Brandacher G, Kumar AR (2017) Vascular endothelial growth factor induction of muscle-derived stem cells enhances vascular phenotype while preserving myogenic potential. Ann Plast Surg 79:404–409

    Article  CAS  Google Scholar 

  • Wu X, Wang S, Chen B, An X (2010) Muscle-derived stem cells: isolation, characterization, differentiation, and application in cell and gene therapy. Cell Tissue Res 340:549–567

    Article  Google Scholar 

  • Xue R, Li JY, Yeh Y, Yang L, Chien S (2013) Effects of matrix elasticity and cell density on human mesenchymal stem cells differentiation. J Orthop Res 31:1360–1365

    Article  CAS  Google Scholar 

  • Zambon JP, de Sa Barretto LS, Nakamura AN, Duailibi S, Leite K, Magalhaes RS, Orlando G, Ross CL, Peloso A, Almeida FG (2014) Histological changes induced by polyglycolic-acid (PGA) scaffolds seeded with autologous adipose or muscle-derived stem cells when implanted on rabbit bladder. Organogenesis 10:278–288

    Article  Google Scholar 

  • Zheng B, Cao B, Crisan M, Sun B, Li G, Logar A, Yap S, Pollett JB, Drowley L, Cassino T, Gharaibeh B, Deasy BM, Huard J, Peault B (2007) Prospective identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol 25:1025–1034

    Article  CAS  Google Scholar 

Download references

Funding

This work was supported by the National Natural Science Foundation of China (81571921 and 81671908), the Union Youth Science and Research Fund (3332015155), the Science Fund of Plastic Surgery Hospital, CAMS, PUMC (Q2015013) and the Innovation Fund of Graduate Students, CAMS, PUMC (2017-1002-1-10).

Author information

Authors and Affiliations

  1. Chinese Academy of Medical Science, Peking Union Medical College, Plastic Surgery Hospital, Beijing, 100041, China

    Zhuqiu Xu, Lu Yu, Haibin Lu, Weifeng Feng, Lulu Chen, Jing Zhou, Xiaonan Yang & Zuoliang Qi

Authors
  1. Zhuqiu Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lu Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haibin Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Weifeng Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lulu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jing Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaonan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Zuoliang Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

ZX and LY performed the biopsy and cells culture, HL and WF took charge of the immunofluorescence and flow cytometry, LC and JZ accomplished Statistical analysis. ZX drafted the manuscript. Co-corresponding authors XY and ZQ designed and guided the study. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Xiaonan Yang or Zuoliang Qi.

Ethics declarations

Conflict of interest

The authors declare they have no competing interests.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xu, Z., Yu, L., Lu, H. et al. A modified preplate technique for efficient isolation and proliferation of mice muscle-derived stem cells. Cytotechnology 70, 1671–1683 (2018). https://doi.org/10.1007/s10616-018-0262-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-018-0262-0

Keywords

Search

Navigation