Regenerative or reparative medicine is a new term which emerged in medicine to refer to renewing of damaged organs by stem cells. Mesenchymal stem cells are multipotent progenitor cells that have the capacity to differentiate into all lineages of mesodermal origin. Mesenchymal stem cells (MSCs) have a good potential for inducing anti-inflammatory responses in inflamed tissues, hence they are used as therapy in auto-inflammatory disease. Due to their differentiation, proliferation and self- renewing characteristics, have received attentions with regard to their potential use as therapeutic agent. This promising area of science is leading scientists to investigate the possibility of cell-based therapies to treat different kinds of diseases, e.g., GVHD, Multiple sclerosis, diabetes and etc. There are two population of cells in the bone marrow that can be differentiated to MSCs in vitro. Mesenchymal stem cells derived from the non-adherent cell population of human bone marrow cell cultures had similar cell proliferation rates in vitro when compared with the MSCs derived from the primary adherent cell population. While there are different methods for isolation and preparation of MSCs from different tissues, but none of them are completely standard. All these methods need additional manipulations of cells, which may affect their differentiation potentials as well as increasing the risk of contamination of cultures. Therefore, there is a need to compare these populations from different aspects. We hypothesize that non adherent MSCs population are more applicable than adherent one, therefore, culturing and manipulating of non-adherent MSCs population is easier than adherent MSCs. In addition, non-adherent MSCs culturing is a cost-effective way as well as increasing the number of cells and shortening the time of the cell culture.

Keywords: mesenchymal stem cell, adherent, non-adherent, cell culture, regenerative or reparative medicine, embryonic stem cells

MSCs, mesenchymal stem cells; iNOS, inducible nitric oxide synthase; COX, cyclooxygenases; PGE2, metabolite prostaglandin e2

It has been many years since scientists desired to regenerate damaged tissues and give them another chance to live. Stem cells are fundamental cells which have the capacity to self-renew and to give rise to cells of various lineages. “Regenerative or reparative medicine” is almost new term which has been entered in medicine refer to renewing of damaged organs by stem cells.¹ Overall, there are two kinds of stem cells in human body; embryonic stem cells and adult stem cells. One type of adult stem cells is mesenchymal stem cells (MSCs). Mesenchymal stem cells are multipotent progenitor cells that have the capacity to differentiating into all lineages of mesodermal origin, e.g., fabricate bone, cartilage, adipose, tendon, muscle, and other connective tissues.^2,3 After the discovery and isolation of adherent mesenchymal stem cells by Friedenstein in 1968,⁴ a gleam of hope appeared which motivate scientist to do more studies on this cell and its biological function. Furthermore, it was discovered that MSCs could be isolated from other tissues such as an adipose tissue, peripheral blood, umbilical cord and placenta which make it more applicable for clinical usage⁵ MSCs are easily isolated from bone marrow and adipose tissue and are expanded in vivo.⁶ MSCs are immune evasive cells which can been transplanted between individuals of the same species which after administration, will migrate to inflamed tissue and by releasing antigenic and trophic substrates, will help to regeneration of damaged tissue.^7–9 In other hand, significant trait of MSCs is their immunomodulatory effect which is proceded by secretion of anti-inflammatory cytokine such as IL-10 and TGF-b.^10,11 In fact, a lot of investigations have revealed that MSC therapy is a worthwhile strategy to down-regulate pathogenic immune responses in graft-versus-host and autoimmune diseases.¹² Interestingly, some scientific literatures suggested that MSCs are categorized in two groups in bone marrow; non-adherent MSC¹³ and adherent MSC. From the beginning, it was acclaimed that non adherent MSCs have similar proliferation and differentiation potentials as the adherent MSCs.¹⁴ This mini-review will provide an overview of the recent findings related to adherent-MSCs and non-adherent MSCs application in clinical therapy.

In 2006, the International Society of Cellular Therapy defined MSCs by different criteria which make it characterization easier.¹⁵ These criteria are; (1) adherent to plastic under standard tissue culture conditions; (2) expressing certain cell surface markers such as CD73, CD90, and CD105, and lack of expression in other markers, including CD45, CD34, CD14, or CD11b, CD79alpha or CD19 and HLA-DR surface molecules and have the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts under vitro conditions.³ There are four Biological characteristics of MSCs associated with their therapeutic effects. These are; Capacity to migrate and engraft, Differentiation, Secreting multiple bioactive molecules and Immunomodulatory functions.^16,17 The therapeutic efficacy of MSCs which is greatly dependent on their ability to produce paracrine factors, will not be effective until they are delivered to site of injury, this process is termed “homing”. Migration and homing to the tissue of injury is influenced by multiple factors including age and passage number of the cells, culture conditions, and the administration method. We here provide a review of the literature demonstrating the effect of various factors on migration of MSCs. Former studies have shown that administrated MSCs could migrate to inflamed sites by chemotaxis.^18,19 These mean that MSCs will arrive to the target site just because of their chemokine receptor such as CCR2, CCR3, CCR4 or CCL5.^20,21 After arriving of MSC in damaged tissue, they can have differentiated into other cells, such as muscle cells, epithelial cell, fibroblast and etc.²² The process which leads to MSCs differentiation is not completely known, but what expected is that various substances and different signals in target place will lead to this differentiation and proliferation. Another significant role of MSCs is their abilities to secrete different molecules which help the remission of inflammation.^23,24 Contrast immunomodulatory effect of MSCs is most due to it soluble factor which release by inflammatory microenvironment stimuli.²⁵ These factors are including; indoleamine 2,3-dioxygenase (IDO), inducible nitric oxide synthase (iNOS), cyclooxygenases (COX), metabolite prostaglandin E2 (PGE2), tumor necrosis factor α-induced protein 6 (TSG6), transforming growth factor β (TGF-β), soluble form of HLA-G5 and IL-10^26–29 studies have shown that MSc by realizing anti-inflammatory cytokine could prevent autoimmunity responses.^30,31 By this, MSCs can reduce the activity of T helper cells,³² B cells³³ and NK cells³⁴ which are the key players in autoimmunity disease. The immunomodulatory effects of MSCs have also been examined in animal models of immune diseases.

The first clinical trial using culture-expanded MSCs was performed in 1995 on 15 patients whom were treated with autologous stem cells.³⁵ Clinical applications of MSCs are evolving with the ambitious goals of improving hematopoietic engraftment rate and pace, ameliorating or preventing GVHD,³⁶ correcting inborn metabolic errors and delivering a variety of therapeutic genes and regeneration of damage tissue in different organ in diverse situation such as IBD.^37,38 As evidences show, most of the studies on allogenic or autogenic MSCs in trials are Phase I studies, Phase II, and a combination of Phase I/II studies. Only a small number of these trials are in Phase III or Phase II/III (comparing a newer treatment to the standard or best known treatment). Nevertheless, adherent MSCs are a wonderful candidate for disease treatment but there is some obstacle in the way of its clinical usage. For instance, isolation and culturing of adherent MSCs is a costly procedure. After isolation, cells should be centrifuged and transferred to a flask and every 2-3 days its medium culture should be changed until the desired number of MSCs are gained (about 19-21 days).³⁹ It has also shown that the number of adherent MSCs in bone marrow is too low, about 1 in 10,000 nucleated cells, thus it means we need more amount of bone marrow cells and subsequently more culture medium, more damage to bone and finally more rate of infection by doing procedures. Additionally, we know that culturing and sub culturing processes are susceptive to bacterial contamination. If we take a precise look at it, we will find out that all of these will impose more charge to the patient. Zhang and et al shows that, non-adherent MSCs are another type of mesenchymal stem cell progenitors which however couldn’t attach to plastic while can differentiate to divers lineage such as chondrocytic and adipocytes and etc.¹⁴ By pour-off’, non-adherent MSCs methods it is feasible to show that non-adherent MSCs are present in bone marrow. Studies show that, non-adherent MSCs have a potential therapeutic effect on the hematopoietic system regeneration and damaged tissue repairing.¹⁴ Mesenchymal stem cells derived from the non-adherent cell population of human bone marrow cell cultures had similar cell proliferation rates in vitro when compared with the MSCs derived from the primary adherent cell population. It has been suggested that the transformation from non-adherent mesenchymal to adherent phenotype might be involved in the actions of bone anabolic drugs such prostaglandin E2 and parathyroid hormone.^40,41 In other study, Stephan Fricke et al demonstrated that non-adherent mesenchymal triggered endogenous hematopoiesis and induced faster recovery compared to bone marrow controls.⁴²

From three decades ago when Friedenstein discovered MSCs in bone marrow specimen, usage of culture-expanded marrow derived MSCs in the fields of tissue engineering, cell therapy, and gene therapy has become popular and widely accepted. Therefore, it needs to use, the safe, effective, and standardized full-scale methods to isolate and culturing MSCs. While there are different methods for isolation and preparation of MSCs from different tissues, but none of them are completely standard. All these methods need additional manipulations of cells, which may affect their differentiation potentials as well as increasing the risk of contamination of cultures. As studies show, the number of non-adherent MSCs in the primary sample of bone marrow is more than adherent MSCs which make it easier for culturing and manipulating. In conclusion, this mini review established that using non adherent MSCs is easier and cost-effective way as well as increasing the number of cells and shortening the time of the cell culture.

None.

The author declares no conflict of interest.

1. Caplan AI. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J Cell Physiol. 2007;213(2):341–347.
2. Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143–147.
3. Wada N, Gronthos S, Bartold PM. Immunomodulatory effects of stem cells. Periodontol. 2013;63(1):198–216.
4. Friedenstein AJ, Petrakova KV, Kurolesova AI, et al. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 1968;6(2):230–247.
5. Xian YF, Lin ZX, Ip SP, et al. Comparison the neuropreotective effect of Cortex Phellodendri chinensis and Cortex Phellodendri amurensis against beta–amyloid–induced neurotoxicity in PC12 cells. Phytomedicine. 2013;20(2):187–193.
6. Scherjon SA, Kleijburg van der Keur C, de Groot Swings GM, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem cells. 2004;22(7):1338–1345.
7. Modo M, Cash D, Mellodew K, et al. Tracking transplanted stem cell migration using bifunctional, contrast agent–enhanced, magnetic resonance imaging. Neuroimage. 2002;17(2):803–811.
8. Zhu H, Mitsuhashi N, Klein A, et al. The role of the hyaluronan receptor CD44 in mesenchymal stem cell migration in the extracellular matrix. Stem cells. 2006;24(4):928–935.
9. Sohni A, Verfaillie CM. Mesenchymal stem cells migration homing and tracking. Stem Cells International. 2013;2013:8.
10. Bartholomew A, Sturgeon C, Siatskas M, et al. Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol. 2002;30(1):42–48.
11. Crop M, Baan C, Weimar W, et al. Potential of mesenchymal stem cells as immune therapy in solid–organ transplantation. Transpl Int. 2009;22(4):365–376.
12. Ghannam S, Bouffi C, Djouad F, et al. Immunosuppression by mesenchymal stem cells:mechanisms and clinical applications. Stem cell Res Ther. 2010;1(1):2.
13. Wan C, He Q, McCaigue M, et al. Nonadherent cell population of human marrow culture is a complementary source of mesenchymal stem cells (MSCs). J Orthop Res. 2006;24(1):21–28.
14. Zhang Z, Tong J, Lu R, et al. Therapeutic potential of non–adherent BM–derived mesenchymal stem cells in tissue regeneration. Bone marrow transplant. 2009;43(1):69–81.
15. 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.
16. Nauta AJ, Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007;110(10):3499–3506.
17. Kassem M. Mesenchymal stem cells: biological characteristics and potential clinical applications. Cloning Stem Cells. 2004;6(4):369–374.
18. Karp JM, Teo GSL. Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell. 2009;4(3):206–216.
19. Spaeth E, Klopp A, Dembinski J, et al. Inflammation and tumor microenvironments: defining the migratory itinerary of mesenchymal stem cells. Gene Ther. 2008;15(10):730–738.
20. Ringe J, Strassburg S, Neumann K, et al. Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2. J Cell Biochem. 2007;101(1):135–146.
21. Lüttichau IV, Notohamiprodjo M, Wechselberger A, et al. Human adult CD34–progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4. Stem Cells Dev. 2005;14(3):329–336.
22. Baksh D, Song L, Tuan R. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med. 2004;8(3):301–316.
23. Bouffi C, Bony C, Courties G, et al. IL–6–dependent PGE2 secretion by mesenchymal stem cells inhibits local inflammation in experimental arthritis. PloS One. 2010;5(12):e14247.
24. Mei SH, Haitsma JJ, Dos Santos CC, et al. Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med. 2010;182(8):1047–1057.
25. Kyurkchiev D, Bochev I, Ivanova Todorova E, et al. Secretion of immunoregulatory cytokines by mesenchymal stem cells. World J Stem Cells. 2014;6(5):552–570.
26. Gao F, Chiu SM, Motan DAL, et al. Mesenchymal stem cells and immunomodulation:current status and future prospects. Cell Death Disease. 2016;7(1):e2062.
27. Krampera M, Cosmi L, Angeli R, et al. Role for interferon–gamma in the immunomodulatory activity of human bone marrow mesenchymal stem cells. Stem cells. 2006;24(2):386–398.
28. Ryan JM, Barry F, Murphy JM, et al. Interferon–gamma does not break, but promotes the immunosuppressive capacity of adult human mesenchymal stem cells. Clin Exp Immunol. 2007;149(2):353–363.
29. DelaRosa O, Lombardo E, Beraza A, et al. Requirement of IFN–gamma–mediated indoleamine 2,3–dioxygenase expression in the modulation of lymphocyte proliferation by human adipose–derived stem cells. Tissue Eng Part A. 2009;15(10):2795–2806.
30. Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response of naive and memory antigen–specific T cells to their cognate peptide. Blood. 2003;101(9):3722–3729.
31. González MA, Gonzalez Rey E, Rico L, et al. Adipose–derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology. 2009;136(3):978–989.
32. Rasmusson I, Ringdén O, Sundberg B, et al. Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells. Transplantation. 2003;76(8):1208–1213.
33. Corcione A, Benvenuto F, Ferretti E, et al. Human mesenchymal stem cells modulate B–cell functions. Blood. 2006;107(1):367–372.
34. Sotiropoulou PA, Perez SA, Gritzapis AD, et al. Interactions between human mesenchymal stem cells and natural killer cells. Stem cells. 2006;24(1):74–85.
35. Lazarus HM, Haynesworth SE, Gerson SL, et al. Ex vivo expansion and subsequent infusion of human bone marrow–derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant. 1995;16(4):557–564.
36. Le Blanc K, Frassoni F, Ball L, et al. Mesenchymal stem cells for treatment of steroid–resistant, severe, acute graft–versus–host disease:a phase II study. The Lancet. 2008;371(9624):1579–1586.
37. García Olmo D, García Arranz M, Herreros D, et al. A phase I clinical trial of the treatment of Crohn’s fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum. 2005;48(7):1416–1423.
38. Barry FP, Murphy JM. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol. 2004;36(4):568–584.
39. Soleimani M, Nadri S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nature protocols. 2009;4(1):102–126.
40. Davies J, Chambers TJ. Parathyroid hormone activates adhesion in bone marrow stromal precursor cells. J Endocrinol. 2004;180(3):505–513.
41. Scutt A, Bertram P. Bone marrow cells are targets for the anabolic actions of prostaglandin E2 on bone: induction of a transition from nonadherent to adherent osteoblast precursors. J Bone Miner Res. 1995;10(3):474–487.
42. Fricke S, Ackermann M, Stolzing A, et al. Allogeneic non–adherent bone marrow cells facilitate hematopoietic recovery but do not lead to allogeneic engraftment. PloS One. 2009;4(7):e6157.