Charachterization of umbilical cord mesenchymal stromal cells during long-term expansion in vitro

Cover Page

Cite item

Full Text

Abstract

One of the clinicians’ major concerns is the biological safety of MSC. The critical question for clinical application of human MSC is their ability to undergo spontaneous malignant transformation in a recipient organism. The goal of our research was to study umbilical cord hMSC proliferative and differentiation capacities, karyotype stability, telomerase activity and telomere length, oncomarkers expression and tumorigenicity during long-term (6 months) cultivation ex vivo. Here we report on the establishing the primary culture of human umbilical cord MSC, MSC_0714, that was capable to proliferate ex vivo for up to 59 passages (6 months). During this period, the cells preserved their normal karyotype, morphology and MSC immunophenotype. Telomeres started to shorten only after the passage 20, while hTERT was inactive in these cells for the whole period of expansion. At the beginning of cultivation the number of SA-β-gal positive cells did not exceeded 3-5%, after the 22th passage their number started to increase and reached 49% at the passage 57. Thus, it was shown that MSC during long-term culture retain their characteristics and undergo cell senescence.

About the authors

A. A. Aizenshtadt

North-Western State Medical University named after I.I. Mechnikov

Author for correspondence.
Email: nie@newmail.ru
Russian Federation, Saint Petersburg

M. A. Skazina

Stem Cell Bank Pokrovsky

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

E. A. Kotelevskaya

Stem Cell Bank Pokrovsky

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

L. V. Yelsukova

Stem Cell Bank Pokrovsky

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

T. L. Zolina

Stem Cell Bank Pokrovsky

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

N. V. Ponomartsev

Institute of Cytology RAS

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

N. K. Galaktionov

Institute of Cytology RAS

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

I. A. Galembo

Stem Cell Bank Pokrovsky

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

D. A. Ivolgin

North-Western State Medical University named after I.I. Mechnikov

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

I. I. Maslennikova

North-Western State Medical University named after I.I. Mechnikov

Email: nie@newmail.ru
Russian Federation, Saint Petersburg, Russia

N. I. Enukashvily

North-Western State Medical University named after I.I. Mechnikov

Email: nie@newmail.ru
Russian Federation, Saint Petersburg

References

  1. 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. Cytotherapy. 2006;8(4):315-317. doi: 10.1080/14653240600855905.
  2. Furno DL, Mannino G, Giuffrida R. Functional Role of Mesenchymal Stem Cells in the Treatment of Chronic Neurodegenerative Diseases. J Cell Physiol. 2018;233(5):3982-3999. doi: 10.1002/jcp.26192.
  3. Epstein SE, Luger D, Lipinski MJ. Paracrine-Mediated Systemic Anti-Inflammatory Activity of Intravenously Administered Mesenchymal Stem Cells: A Transformative Strategy for Cardiac Stem Cell Therapeutics. Cir Res. 2017;121(9):1044-1046. doi: 10.1161/CIRCRESAHA.117.311925.
  4. Mohseny AB, Szuhai K, Romeo S, et al. Osteosarcoma originates from mesenchymal stem cells in consequence of aneuploidization and genomic loss of Cdkn2. J Pathol. 2009;219(3):294-305. doi: 10.1002/path.2603.
  5. Torsvik A, Røsland GV, Svendsen A, et al. Spontaneous malignant transformation of human mesenchymal stem cells reflects cross-contamination: putting the research field on track-letter. Cancer Res. 2010;70(15):6393-6396. doi: 10.1158/0008-5472.CAN-10-1305.
  6. Tao H, Lin Y, Zhang G, et al. Experimental observation of human bone marrow mesenchymal stem cell transplantation into rabbit intervertebral discs. Biomed Rep. 2016;5(3):357-360. doi: 10.3892/br.2016.731.
  7. Katsiani E, Garas A, Skentou C, et al. Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing. Cell and Tissue Banking. 2016;17(3):517-529. doi: 10.1007/s10561-016-9559-4.
  8. Jeong SG, Cho GW. Accumulation of apoptosis-insensitive human bone marrow-mesenchymal stromal cells after long-term expansion. Cell Biochemistry and Function. 2016;34(5):310-316. doi: 10.1002/cbf.3191.
  9. Danisovic L, Oravcova L, Krajciova L, et al. Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem cells. J Physiol Pharmacol. 2017;68(1):149-158.
  10. Nikitina V, Astrelina T, Nugis V, et al. Clonal chromosomal and genomic instability during human multipotent mesenchymal stromal cells long-term culture. PLoS One. 2018;13(2):e0192445. doi: 10.1371/journal.pone.0192445.
  11. Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 2007;25(6):1384-1392. doi: 10.1634/stemcells.2006-0709.
  12. Айзенштадт А.А., Енукашвили Н.И., Золина Т.Л., и др. Сравнение пролиферативной активности и фенотипа МСК, полученных из костного мозга, жировой ткани и пупочного канатика // Вестник Северо-Западного государственного медицинского университета им. И.И. Мечникова. – 2015. – Т. 7. – № 2. – С. 14–22. [Aisenstadt AA, Enukashvili NI, Zolina TL. Comparison of proliferation and immunophenotype of MSK, obtainedfrom bone marrow, adipose tissue and umbilical cord. Herald of North-Western State Medical University named after I.I. Mechnikov. 2015;7(2):14-22. (In Russ.)]
  13. Ma YH, Zeng X, Qiu XC, et al. Perineurium-like sheath derived from long-term surviving mesenchymal stem cells confers nerve protection to the injured spinal cord. Biomaterials. 2018;160:37-55. doi: 10.1016/j.biomaterials.2018.01.015.
  14. Wang Y, Zhang Z, Chi Y, et al. Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation. Cell Death Dis. 2013;4(12):e950. doi: 10.1038/cddis.2013.480.
  15. Hao H, Chen G, Liu J, et al. Culturing on Wharton’s jelly extract delays mesenchymal stem cell senescence through p53 and p16INK4a/pRb pathways. PLoS One. 2013;8(3):e58314. doi: 10.1371/journal.pone.0058314.
  16. Kundrotas G, Gasperskaja E, Slapsyte G, et al. Identity, proliferation capacity, genomic stability and novel senescence markers of mesenchymal stem cells isolated from low volume of human bone marrow. Oncotarget. 2016;7(10):10788-802. doi: 10.18632/oncotarget.7456.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Average time doubling of the population of mesenchymal stromal cells of umbilical cord. The blue line is the mean time of doubling the population of mesenchymal stromal cells in 204 cultures. The red line is the time of doubling the population of mesenchymal stromal cells _0714. The abscissa is the number of passages, the ordinate is the time of population doubling. The data are presented as an average value with a deferred standard deviation

Download (78KB)
Indexing metadata
3. Fig. 2. Representative micrographs of culture cells MSC_0714 in early (4th passage), medium (22nd passage) and late (47th passage) cultivation times. Light microscopy. Uv. ×400. The scale (50 μm) is indicated in micrographs

Download (434KB)
Indexing metadata
4. Fig. 3. Culture MSC_0714 on the 48th passage. Blue — cells with active SA-β-gal. Light microscopy. Uv. ×400. The scale is indicated in micrographs

Download (255KB)
Indexing metadata
5. Fig. 4. Intensity of fluorescence of culture cells MSC_0714, expressing antigens CD90 and CD105 as a function of passage

Download (230KB)
Indexing metadata
6. Fig. 5. Type of cultures MSC_0714 of the 28th passage after the second week of cultivation in a medium containing chondrogenic (a — staining with alcian blue), adipogenic (b — staining with fatty red O) and osteogenic stimuli (c — staining with alizarin red). Light microscopy. Uv. ×400. The scale is indicated in micrographs

Download (513KB)
Indexing metadata
7. Fig. 6. Change in telomere length of cell cultures of MSK_0714 during in vitro culture

Download (58KB)
Indexing metadata
8. Fig. 7. Activity of telomerase in the cells of MSK_0714. KV and 1301 — positive control, cells with high-activity telomerase hTERT

Download (34KB)
Indexing metadata
9. Fig. 8. Karyotype MSC_0714 at 5 months after the beginning of cultivation (50th passage)

Download (366KB)
Indexing metadata

Copyright (c) 2018 Aizenshtadt A.A., Skazina M.A., Kotelevskaya E.A., Yelsukova L.V., Zolina T.L., Ponomartsev N.V., Galaktionov N.K., Galembo I.A., Ivolgin D.A., Maslennikova I.I., Enukashvily N.I.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies