TSitologiya i Genetika 2022, vol. 56, no. 2, 38-48
Cytology and Genetics 2022, vol. 56, no. 2, 139–147, doi: https://www.doi.org/10.3103/S0095452722020098

Neuroregenerative «bystander»-effects of conditioned media from adipose tissue-derived fibroblast-like cells in vitro

Pedachenko E.G., Liubich L.D., Staino L.P., Egorova D.M., Skaterna T.D.

  1. The State Institution «Romodanov Neurosurgery Institute, the National Academy of Medical Sciences of Ukraine
  2. Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine

SUMMARY. The use of different types of stem cells, including me-senchymal multipotent stromal cells (MMSCs) and neurogenic stem/progenitor cells (NSCs/NPCs), is a promising direction in the development of new technologies for the treatment of central nervous system diseases. An alternative to direct cell transplantation may be the use of their conditioned media (CM) as a source of secretome and a key component of the mechanism to realize their potential. Current clinical trials, using cell therapy in case of CNS pathology, mostly use adipose tissue, bone marrow, umbilical cord, and cord blood as a source of isolating MMSCs or mononuclear cells of the stromal-vascular fraction, and the lines of immortalized neurogenic cells, isolated from structures of the brain or spinal cord of the human embryo, – as a source of NSCs/NPCs. In experimental conditions, in particular in rodents, adipose tissue is among the most available sources of allogenic progenitor cells of the mesenchymal type, and fetal brain is an accessible source of neurogenic cells. The aim was to study the neuroregenerative effects of conditioned media from rat adipose tissue-derived fibroblast-like cells and fetal neurogenic cells in vitro. CM from 24-h cultures of rat adipose tissue-derived fibroblast-like cells (ad-FLCs) and fetal neurogenic cells (NCs, E14) were examined using electrophoresis in 10 % polyacrylamide gel. On day 5–7, in 2D cultures of rat neural cells (E14) the «scratch assay» was performed and nutrient medium DMEM with 10 % FCS (standard culture conditions, control) or 0.10 mg/ml (by amount of protein) ad-FLCs CM or NCs CM were added. Microscopic and morphometric studies were performed during four days of cultivation. After mechanical transection in the culture of neural cells under standard conditions from the first to the third day there were processes of endogenous regeneration, which decreased to the fourth day. The addition of ad-FLCs CM or NCs CM contributed to a significant increase in the degree and duration of endogenous regeneration processes in neural cell culture. Under the exposure to ad-FLCs CM up to the fourth day, the number of migrated cells and the density of cell processes in the scratch area increased 7 and 12.5 times, respectively; the exposure to NCs CM increased the number of migrated cells (3.5 times), the distance of cell migration (1.4 times), the density of cell processes (13 times). The length of the overgrown section of the scratch area increased 1.7 times after the exposure to ad-FLCs CM, NCs CM – 3 times, amounting to 23.7 and 43.5 % respectively of the total length of the transection zone. 10 protein fractions were detected in the ad-FLCs CM: predominant 12, 15, 23, 30, 80 kDa and minor – 28, 35, 55, 65, 75 kDa; in NCs CM – 9 fractions: prevailing 15, 23, 30, 35 kDa and minor – 37, 40, 46, 67, 80 kDa. CM from 24-h cultures of rat ad-FLCs or NCs (E14) stimulate endogenous regeneration processes in rat brain cell culture with mechanical monolayer transection. The affecting factors of neuroregenerative «bystander» effects of rat ad-FLCs or NCs are secreted biologically active proteins – components of the CM predominant and minor protein fractions.

Keywords: conditioned medium, adipose tissue-derived fibroblast-like cells, neurogenic cells, scratch assay, electrophoresis, protein fractions

TSitologiya i Genetika
2022, vol. 56, no. 2, 38-48

Current Issue
Cytology and Genetics
2022, vol. 56, no. 2, 139–147,
doi: 10.3103/S0095452722020098

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References

Bao, X., Feng, M., Wei, J. et al., Transplantation of Flk-1+ human bone marrow-derived mesenchymal stem cells promotes angiogenesis and neurogenesis after cerebral ischemia in rats, Eur. J. Neurosci., 2011, vol. 34, pp. 87–98. https://doi.org/10.1111/j.1460-9568.2011.07733.x

Beretta, S., Cunningham, K.M., Haus, D.L., et al., Effects of human ES-derived neural stem cell transplantation and kindling in a rat model of traumatic brain injury, Cell Transplant., 2017, vol. 26, pp. 1247–1261. https://doi.org/10.1177/0963689717714107

Cunningham, C.J., Enrich, M.V., Pickford, M.M. et al., The therapeutic potential of the stem cell secretome for spinal cord repair: a systematic review and meta-analysis, OBM Neurobiol., 2020, vol. 4, no. 4. https://doi.org/10.21926/obm.neurobiol.2004080

Dobrowolski, S. and Lepski, G., Stem cells in traumatic brain injury, Am. J. Neurosci., 2013, vol. 4, pp. 13–24. https://doi.org/10.3844/ajnsp.2013.13.24

Gao, J., Grill, R.J., Dunn, T.J., et al., Human neural stem cell transplantation-mediated alteration of microglial/macrophage phenotypes after traumatic brain injury, Cell Transplant., 2016, vol. 25, pp. 1863–1877. https://doi.org/10.3727/096368916X691150

Gomazkov, O.A., Neurogenesis as an adaptive function of the brain, Biol. Bull. Rev., 2014, vol. 4, pp. 86–100.

Haus, D.L., López-Velázquez, L., Gold, E.M., et al., Transplantation of human neural stem cells restores cognition in an immunodeficient rodent model of traumatic brain injury, Exp. Neurol., 2016, vol. 281, pp. 1–16. https://doi.org/10.1016/j.expneurol.2016.04.008

Kocan, B., Maziarz, A., Tabarkiewicz, J., et al., Trophic activity and phenotype of adipose tissue-derived mesenchymal stem cells as a background of their regenerative potential, Stem Cells Int., 2017, vol. 2017, art. ID 1653254. https://doi.org/10.1155/2017/1653254

Kopach, O. and Pivneva, T., Cell-based therapies for neural replacement strategies in stroke-related neurodegeneration: neurophysiological insights into stem progenitor cell neurogenesis within a host environment, Neural Regener. Res., 2018, vol. 13, pp. 1350–1351. https://doi.org/10.4103/1673-5374.235224

Lee, M.C., Jin, C.Y., Kim, H.S., et al., Stem cell dynamics in an experimental model of stroke, Chonnam Med. J., 2011, vol. 47, no. 2, pp. 90–98. https://doi.org/10.4068/cmj.2011.47.2.90

Ludwig, P.E., Thankam, F.G., Patil, A.A., et al., Brain injury and neural stem cells, Neural Regener. Res., 2018, vol. 13, no. 1, pp. 7–18. https://doi.org/10.4103/1673-5374.224361

Qu, X. and Sheng, H., Stem cell therapy for traumatic brain injury: a progress update, Ann. Neurol. Surg., 2018, vol. 2, art. ID 1008.

Semenova, V., Lisianyi, N., Stayno, L., et al., Proliferative and differentiated potential of mesenchymal stem cells from adipose tissue under cultivation conditions, Ukr. Neurosurg. J., 2014, vol. 3, pp. 24–29 https://doi.org/10.25305/unj.47487

Shi, W., Huang, C.J., Xu, X.D., et al., Transplantation of RADA16-BDNF peptide scaffold with human umbilical cord mesenchymal stem cells forced with CXCR4 and activated astrocytes for repair of traumatic brain injury, Acta Biomater., 2016, vol. 45, pp. 247–261. https://doi.org/10.1016/j.actbio.2016.09.001

Walker, P.A., Letourneau, P.A., Bedi, S., et al., Progenitor cells as remote “bioreactors”: Neuroprotection via modulation of the systemic inflammatory response, World J. Stem Cells, 2011, vol. 3, pp. 9–18. https://doi.org/10.4252/wjsc.v3.i2.9

Webb, R.L., Kaiser, E.E., Spellicy, S., et al., Human neural stem cell extracellular vesicles improve tissue and functional recovery in the murine thromboembolic stroke model, Transl. Stroke Res., 2018, vol. 9, pp. 530–539. https://doi.org/10.1007/s12975-017-0599-2

Wei, L., Fraser, J.L., Lu, Z.Y., et al., Transplantation of hypoxia preconditioned bone marrow mesenchymal stem cells enhances angiogenesis and neurogenesis after cerebral ischemia in rats, Neurobiol. Dis., 2012, vol. 46, no. 3, pp. 635–645. https://doi.org/10.1016/j.nbd.2012.03.002

Willis, C.M., Nicaise, A.M., Hamel, R., et al., Harnessing the neural stem cell secretome for regenerative neuroimmunology, Front. Cell Neurosci., 2020, vol. 14, art. ID 590960. https://doi.org/10.3389/fncel.2020.590960

Xu, Ch., Diao, Y.F., Wang, J., et al., Intravenously infusing the secretome of adipose-derived mesenchymal stem cells ameliorates neuroinflammation and neurological functioning after traumatic brain injury, Stem Cells Dev., 2020, vol. 29, pp. 222–234. https://doi.org/10.1089/scd.2019.0173

Yang, H., Wang, C., Chen, H., et al., Neural stem cell-conditioned medium ameliorated cerebral ischemia-reperfusion injury in rats, stem cells international, 2018, vol. 2018, art. ID 4659159. https://doi.org/10.1155/2018/4659159

Zhong, D., Cao, Y., Li, C.J., et al., Neural stem cell-derived exosomes facilitate spinal cordfunctional recovery after injury by promoting angiogenesis, Exp. Biol. Med., 2020, vol. 245, no. 1, pp. 54–65. https://doi.org/10.1177/1535370219895491