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 Table of Contents    
ORIGINAL ARTICLE
Year : 2018  |  Volume : 51  |  Issue : 2  |  Page : 177-181
 

Adipose tissue: A natural resource for multipotent mesenchymal stem cells with potential translation to trigerminal layers


1 Samanvaya Trust, Vadodara, Gujarat, India
2 Manipal Institute of Regenerative Medicine, Bengaluru, Karnataka, India

Date of Web Publication29-Oct-2018

Correspondence Address:
Dr. Rajni Vyas
Parimal Pratap Road, Raopura, Vadodara - 390 001, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijps.IJPS_150_17

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 » Abstract 

Background: The article reports basic science research that establishes that adipose tissue (AT)-derived mesenchymal stem cells (MSCs) have a potential to transgerminal translation. Study Design: MSC confirmation was obtained by phenotypic spindle-shaped cells as well as with four positive and three negative markers. The translineage translation of adipose-derived MSCs (ADMSCs) was established. Materials and Methods: The lipoaspirate was subjected to enzymatic digestion with collagenase. Stromal vascular factor (SVF) was isolated. With two passages, pure culture of ADMSCs was obtained. They were translated to all the three germinal layers. Results: AT-derived SVF contains ~30% MSCs. They are capable of being translated into endoderm, mesoderm and ectoderm. Conclusion: AT is a rich source for MSCs, with immense research possibilities for regeneration and rejuvenation.


Keywords: Adipose tissue; mesenchymal stem cells; stromal vascular fraction


How to cite this article:
Vyas B, Shah A, Marathe A, Ansarullah, Vyas R, Bhonde R. Adipose tissue: A natural resource for multipotent mesenchymal stem cells with potential translation to trigerminal layers. Indian J Plast Surg 2018;51:177-81

How to cite this URL:
Vyas B, Shah A, Marathe A, Ansarullah, Vyas R, Bhonde R. Adipose tissue: A natural resource for multipotent mesenchymal stem cells with potential translation to trigerminal layers. Indian J Plast Surg [serial online] 2018 [cited 2018 Dec 12];51:177-81. Available from: http://www.ijps.org/text.asp?2018/51/2/177/244348



 » Introduction Top


Mesenchymal stem cells (MSCs) are adult non-hematopoietic multipotent stem cells. They have received considerable importance in cellular therapy in degenerative disorders,[1],[2] traumatic injuries[3],[4],[5] and autoimmune disorders.[6] They have also been tried in metabolic disorders such as diabetes[7],[8] due to their multiple modes of action and their transgerminal translation potential. They hone to the sight of injury or lesion,[9] they are immunonaïveand in addition, they have immunomodulatory action.[10] In this study, we have gathered evidence of trigerminal translation potential of MSCs in in vitro condition in our GMP Class V, Department of Scientific and Industrial Research approved stem cell research laboratory. This will foster newer modalities of cellular treatment of various disorders.

MSCs are found in varying quantities in the stromal vascular factor (SVF) obtained from different tissues. Adipose tissue (AT) is the richest source for MSCs since SVF obtained therefrom contains approximately 30% of MSCs.[11] This is likely to open up a vast panorama of various conditions that can be treated with cellular products derived from AT.


 » Materials and Methods Top


Selection of subjects for lipoaspiration

AT was obtained from healthy subjects undergoing liposuction for cosmetic reasons at a planned procedure. Informed consent of the subjects willing to contribute the lipoaspirate for research was taken before procedure. All necessary approvals were taken with Institutional Ethics Committee and Institutional Committee for Stem Cell Research and Therapy.

Lipoaspiration

Twenty-five subjects who consented underwent lipoaspiration by the plastic surgeon. A patient was given local anaesthesia. 1–2 cm-long incision was made on abdomen ventrolaterally. Lipoaspirate was taken along with the injected ringer solution under vacuum pressure. Five hundred millilitres lipoaspirate was collected for research purpose from each subject. The lipoaspirate was uniform in consistency and was devoid of any clumps.

Isolation and characterisation of stem cells

Lipoaspirate was washed with phosphate-buffered saline (PBS). Lipoaspirate was then subjected to enzymatic digestion (collagenase type 1, 0.75%) for 2 h with manual shaking. Finally, the aspirate was centrifuged at 1000 g to pellet down the mononuclear cells (MNCs). The MNCs obtained are SVF. The pellet was then washed twice with PBS. 2000 cells/cm2 were seeded in the flask for culturing with Dulbecco's Modified Eagle Medium (DMEM) (HG) and 10% of foetal bovine serum. The cells, adherent to the surface of flask, were used for characterisation using immunocytochemistry (ICC) and flow cytometry. Flow cytometry results were confirmed at two laboratories. ICC imaging of CD44, CD90, CD105, CD34, CD45 and human leucocyte antigen – -antigen D related (HLA-DR) was done using MSC maker kit (Abcam, USA) following manufacturer's instruction. Similarly, quantification of stem cell population was done with flow cytometry using CD 44, CD 90, CD34 and CD45 markers.

Transdifferentiation into trigerminal cell types

MSCs obtained were subjected to differentiation into neurogenic cells (ectoderm), chondrocyte (mesoderm) and islet-like cell aggregates ICAs (endoderm). Neural differentiation was done using commercially available differentiation media (Gibco, Thermo scientific, USA) according to manufacturer's instruction. Similarly, chondrocyte differentiation was done using chondrogenesis media (Gibco, Thermo Scientific, USA). ICAs were generated according to Chandra, 2012 protocol,[12] briefly the confirmed adipose-derived MSCs were plated to low attachment plates (HiMedia Laboratories, Mumbai) at density of 106 cells/cm2 in DMEM: F12 until 80% confluence is achieved. Thereafter, cells were put in serum-free media (SFM) according to the protocol. Differentiation was carried out in three stages; initially, cells were resuspended in SFM-A and were cultured in this media for 2 days. On the 3rd day, cells were shifted to SFM-B media containing taurine. The cell aggregates were cultured in this medium for another 2 days. Finally, cells were further shifted to SFM-C on the 5th day for 14 days. The cell aggregates were fed with fresh SFM-C medium every 2 days in this period.


 » Results Top


Two millilitres of lipoaspirate yielded approximately 1 million SVF cells. SVF was found to contain ~30% MSCs [Figure 1], The figure also reveals cells positive for CD90(31.18%), CD34(12.58%) and CD105(1.37%). In addition, SVF contained an endothelial precursor cell, T-regulatory cells, macrophages, smooth muscle cells, pericytes and pre-adipocytes.
Figure 1: Stromal vascular factor containing CD 90 cells – mesenchymal stem cells

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Cultured and purified MSCs were confirmed with four positive markers CD29, CD44, CD90 and CD105 and were negative for three markers CD34, CD45, CD31 and HLA-DR12. Their translineage translation to mesoderm was confirmed, by evidence for chondrogenesis [Figure 2]. Endodermal translation was confirmed by obtaining insulin-secreting cells [Figure 3]. Ectodermal translation was established with a phenotypic translation to a neuronal-like cell [Figure 4].
Figure 2: Adipose-derived mesenchymal stem cells translated to chondrogenesis

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Figure 3: Adipose-derived mesenchymal stem cells translated to islet-like cell aggregates

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Figure 4: Adipose-derived mesenchymal stem cells translated to neuronal-like cells

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 » Discussion Top


The cost of finding a new molecular drug has been steadily increasing from $1.2 billion in 2001 to ~$3.0 billion in 2017 (Pfizer company report).

MSCs derived from bone marrow underwent extensive research and application for regeneration. In the present decade, this cell line is being replaced by yet another easily accessed and more abundant source of MSCs – this is AT. This study reflects on a huge potential for plastic surgeons to utilise the skills to extend to a super speciality of regenerative medicine as an autologous source. The safety of MSCs by intra-articular, intramuscular and intravenous route is established.[13] The dosages range from as low as 10 million MSCs[13] to 5 million MSCs/kg body weight (BW).[14],[15] We have exhibited 3–4 million/kg BW SVF cells.[16]

The human AT is an abundant source for stromal vascular fraction, containing a cocktail of mesenchymal as well as haemopoiesis, and other cells provide a base for cell therapy. Thus, it has established its place as a plausible source for therapeutic application (PRS 2015).

We have proven the therapeutic application of SVF with platelet-rich plasma for osteoarthritis of the knee.[16] Osteoarthritis anywhere else in the body has similar pathogenesis and morbid anatomy with only minor variations; therefore, we believe that SVF will have therapeutic applications there also. SVF with enrichment of platelets can also be applied to avascular necrosis of femur,[17] and even to delayed healing at fracture sites.[18] A further research possibility exists for application of SVF in rheumatoid joints as well.

Besides this, researchers have exhibited SVF to treat extensive burns,[19] diabetic foot,[20] breast augmentation,[21] nerve regeneration[22] and wound healing,[23] diabetes[24] and ischemic heart failure.[25]

MSC translation to insulin-producing cells has a potential for application for type I and II diabetes.[12] We have proven glucose-lowering activity of SVF in seven diabetic patients.[26] MSCs have research potential for application to several neurological disorders that may range from children suffering from cerebral palsy[27] to adults suffering from syringomyelia,[28] Parkinson's disease,[29] Alzheimer's disease[30] etc.


 » Conclusion Top


The study adds the evidence for translineage translation of MSCs obtained from AT to existing literature. This opens up a wide panorama for research in therapeutic applications for unmatched challenges. The study also puts a product in the palm of the plastic surgeon available at the doorstep of the operating room.

Acknowledgement

Dr. Ramesh Bhonde, the then dean of Manipal Institute of Regenerative Medicine, Bangalore, now the research director at D. Y. Patil, Medical College, Pune, provided mentorship. Dr. Prof. (late) Bharat Chattoo, Head Genomic Research Centre, MSU Vadodara, provided the facility for confocal microscopic studies. Toprani Advance Laboratory systems facilitated with flow cytometry studies. We are grateful to them.

Financial support and sponsorship

The research was partly funded through a SBIRI grant by DBT, Government of India.

Conflicts of interest

There are no conflicts of interest.

 
 » References Top

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Orozco L, Munar A, Soler R, Alberca M, Soler F, Huguet M, et al. Treatment of knee osteoarthritis with autologous mesenchymal stem cells: A pilot study. Transplantation 2013;95:1535-41.  Back to cited text no. 1
    
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Jang WG, Kim EJ, Park KG, Park YB, Choi HS, Kim HJ, et al. Glucocorticoid receptor mediated repression of human insulin gene expression is regulated by PGC-1alpha. Biochem Biophys Res Commun 2007;352:716-21.  Back to cited text no. 2
    
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Mackay-Sim A, Féron F, Cochrane J, Bassingthwaighte L, Bayliss C, Davies W, et al. Autologous olfactory ensheathing cell transplantation in human paraplegia: A 3-year clinical trial. Brain 2008;131:2376-86.  Back to cited text no. 3
    
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Park JH, Kim DY, Sung IY, Choi GH, Jeon MH, Kim KK, et al. Long-term results of spinal cord injury therapy using mesenchymal stem cells derived from bone marrow in humans. Neurosurgery 2012;70:1238-47.  Back to cited text no. 4
    
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et al. Gazdic M, Volarevic V, Harrell CR, Fellabaum C, Jovicic N, Arsenijevic N, et al. Stem cells therapy for spinal cord injury. Int J Mol Sci 2018;19. pii: E1039.  Back to cited text no. 5
    
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Le Blanc K, Rasmusson I, Sundberg B, Götherström C, Hassan M, Uzunel M, et al. Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 2004;363:1439-41.  Back to cited text no. 6
    
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Wu H, Mahato RI. Mesenchymal stem cell-based therapy for type 1 diabetes. Discov Med 2014;17:139-43.  Back to cited text no. 7
    
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Zang L, Hao H, Liu J, Li Y, Han W, Mu Y, et al. Mesenchymal stem cell therapy in type 2 diabetes mellitus. Diabetol Metab Syndr 2017;9:36.  Back to cited text no. 8
    
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Rustad KC, Gurtner GC. Mesenchymal stem cells home to sites of injury and inflammation. Adv Wound Care (New Rochelle) 2012;1:147-52.  Back to cited text no. 9
    
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Nauta AJ and Fibbe WE. Immunomodulatory properties of mesenchymal stromal cells. Blood 2007;110:3499-506.  Back to cited text no. 10
    
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Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adipose-derived stem cells: Isolation, expansion and differentiation. Methods 2008;45:115-20.  Back to cited text no. 11
    
12.
Chandra V, Swetha G, Muthyala S, Jaiswal AK, Bellare JR, Nair PD, et al. Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice. PLoS One 2011;6:e20615.  Back to cited text no. 12
    
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Strauer BE, Brehm M, Zeus T, Gattermann N, Hernandez A, Sorg RV, et al. Intracoronary, human autologous stem cell transplantation for myocardial regeneration following myocardial infarction. Dtsch Med Wochenschr 2001;126:932-8.  Back to cited text no. 13
    
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Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, et al. Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol 2004;94:92-5.  Back to cited text no. 14
    
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Hare JM, Traverse JH, Henry TD, Dib N, Strumpf RK, Schulman SP, et al. A randomized, double-blind, placebo-controlled, dose-escalation study of intravenous adult human mesenchymal stem cells (prochymal) after acute myocardial infarction. J Am Coll Cardiol 2009;54:2277-86.  Back to cited text no. 15
    
16.
Kshatriya P, Marathe A, Thadani J, Vakodikar S, Vyas R, Bhonde R. Innovation of a novel therapeutic modality for treatment of osteoarthritis of knee with autologous adipose derived stromal vascular fraction. IP J Indian Orthop Rheumatol Assoc 2017;3:59-66.  Back to cited text no. 16
    
17.
Pak J, Lee JH, Jeon JH, Lee SH. Complete resolution of avascular necrosis of the human femoral head treated with adipose tissue-derived stem cells and platelet-rich plasma. J Int Med Res 2014;42:1353-62.  Back to cited text no. 17
    
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Valencia Mora M, Ruiz Ibán MA, Díaz Heredia J, Barco Laakso R, Cuéllar R, García Arranz M, et al. Stem cell therapy in the management of shoulder rotator cuff disorders. World J Stem Cells 2015;7:691-9.  Back to cited text no. 18
    
19.
Atalay S, Coruh A, Deniz K. Stromal vascular fraction improves deep partial thickness burn wound healing. Burns 2014;40:1375-83.  Back to cited text no. 19
    
20.
Han SK, Kim HR, Kim WK. The treatment of diabetic foot ulcers with uncultured, processed lipoaspirate cells: A pilot study. Wound Repair Regen 2010;18:342-8.  Back to cited text no. 20
    
21.
Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, Harii K, et al. Cell-assisted lipotransfer for cosmetic breast augmentation: Supportive use of adipose-derived stem/stromal cells. Aesthetic Plast Surg 2008;32:48-55.  Back to cited text no. 21
    
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di Summa PG, Kingham PJ, Raffoul W, Wiberg M, Terenghi G, Kalbermatten DF, et al. Adipose-derived stem cells enhance peripheral nerve regeneration. J Plast Reconstr Aesthet Surg 2010;63:1544-52.  Back to cited text no. 22
    
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Cianfarani F, Toietta G, Di Rocco G, Cesareo E, Zambruno G, Odorisio T, et al. Diabetes impairs adipose tissue-derived stem cell function and efficiency in promoting wound healing. Wound Repair Regen 2013;21:545-53.  Back to cited text no. 23
    
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Jarajapu YP, Grant MB. The promise of cell-based therapies for diabetic complications: Challenges and solutions. Circ Res 2010;106:854-69.  Back to cited text no. 24
    
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You HJ, Han SK. Cell therapy for wound healing. J Korean Med Sci 2014;29:311-9.  Back to cited text no. 25
    
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Vyas B, Vyas R, Ansarullah, Marathe A, Thadani J, Kshatriya P, et al. Therapeutic application of autologous adipose derived mesenchymal stem cells and islet like cell aggregates derived from them in type 1 and 2 diabetes mellitus – A proof of concept study. J Stem Cell Res Dev Ther 2016;3: 008.  Back to cited text no. 26
    
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Zhang C, Huang L, Gu J, Zhou X. Therapy for cerebral palsy by human umbilical cord blood mesenchymal stem cells transplantation combined with basic rehabilitation treatment: A Case report. Glob Pediatr Health 2015;2:2333794x15574091.  Back to cited text no. 27
    
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