Higher Pericyte Content and Secretory Activity of Microfragmented Human Adipose Tissue Compared to Enzymatically Derived Stromal Vascular Fraction

Bianca Vezzani, Isaac Shaw, Hanna Lesme, Li Yong, Nusrat Khan, Carlo Tremolada, Bruno Péault, Bianca Vezzani, Isaac Shaw, Hanna Lesme, Li Yong, Nusrat Khan, Carlo Tremolada, Bruno Péault

Abstract

Autologous adipose tissue is used for tissue repletion and/or regeneration as an intact lipoaspirate or as enzymatically derived stromal vascular fraction (SVF), which may be first cultured into mesenchymal stem cells (MSCs). Alternatively, transplant of autologous adipose tissue mechanically fragmented into submillimeter clusters has recently showed remarkable efficacy in diverse therapeutic indications. To document the biologic basis of the regenerative potential of microfragmented adipose tissue, we first analyzed the distribution of perivascular presumptive MSCs in adipose tissue processed with the Lipogems technology, observing a significant enrichment in pericytes, at the expense of adventitial cells, as compared to isogenic enzymatically processed lipoaspirates. The importance of MSCs as trophic and immunomodulatory cells, due to the secretion of specific factors, has been described. Therefore, we investigated protein secretion by cultured adipose tissue clusters or enzymatically derived SVF using secretome arrays. In culture, microfragmented adipose tissue releases many more growth factors and cytokines involved in tissue repair and regeneration, noticeably via angiogenesis, compared to isogenic SVF. Therefore, we suggest that the efficient tissue repair/regeneration observed after transplantation of microfragmented adipose tissue is due to the secretory ability of the intact perivascular niche. Stem Cells Translational Medicine 2018;7:876-886.

Keywords: Adipose stem cell; Cytokine; Mesenchymal stem cell; Pericyte; Secretome.

© 2018 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Figures

Figure 1
Figure 1
Vasculature in unprocessed and microfragmented adipose tissue. (A, B, C): Endothelial cells are stained with UEA‐1. From left to right: microfragmented adipose tissue (MAT), lipoaspirate (LPA), adipose tissue (AT). Larger vessels were observed only in LPA and AT. (D, E, F): Boxed areas in A, B, C are showed enlarged in D, E, F respectively. Arrowheads indicate pericytes, which have been stained using antibodies against PDGFRβ and NG2. Scale bar: 50 μm.
Figure 2
Figure 2
Respective abundances of perivascular adventitial cells and pericytes in MAT and LPA. (A, B): Dotplots showing adventitial cells (CD34+CD146−) and pericytes (CD146+CD34−) populations in MAT an LPA from the same donor. (C): Quantitative distribution of pericytes and adventitial cells in LPA and MAT, n = 10, *p < .05.
Figure 3
Figure 3
Angiogenic protein secretion by cultured MAT and isogenic SVF. (A): MAT and SVF cultured in basal medium. Scale bar: 500 μm. (B): Angiogenesis proteomic array showing secreted proteins from MAT and SVF after 8 days in culture. Capture antibodies are spotted in duplicate, each dot doublet represents a detected protein. (C): Secretion level of different angiogenic proteins measured as the average of the pixel intensity of the doublets and normalized to the negative control. Statistical analysis was performed on pooled secretion values detected in four separate donors. *p < .05; **p < .01; ***p < .001.
Figure 4
Figure 4
Cytokine secretion by cultured MAT and isogenic SVF. (A): Cytokine proteomic array showing secreted proteins from MAT and SVF after 8 days in culture. Capture antibodies are spotted in duplicate, each dot doublet represent a detected protein. (B): Secretion level of different cytokines measured as the average of the pixel intensity of the doublets and normalized on the negative control. Statistical analysis was performed on pooled secretion values detected in four separate donors. *p < .05; **p < .01; ***p < .001.
Figure 5
Figure 5
Angiogenic protein secretion levels of MAT compared to SVF. Secretion levels of angiogenic factors detected using the angiogenesis proteomic array, expressed as fold change between MAT and SVF values. The values represent the normalized pixel intensity detected in four separate donors. The numbers at the side of the list indicate fold changes.
Figure 6
Figure 6
Cytokine secretion levels of MAT compared to SVF. Secretion levels of cytokines detected using the cytokine proteomic array, expressed as fold change between MAT and SVF values. The values represent the normalized pixel intensity detected in four separate donors. The numbers at the side of the list indicate fold changes.
Figure 7
Figure 7
Protein secretion of enzymatically digested MAT and undigested MAT. (A): Angiogenesis proteomic array and (B) cytokine proteomic array showing secreted proteins from enzymatically digested MAT and undigested MAT after 8 days in culture in basal medium. Capture antibodies are spotted in duplicate, each dot doublet represent a detected protein.

References

    1. Strong AL, Cederna PS, Rubin P et al. The current state of fat grafting: A review of harvesting, processing, and injection techniques. Plast Reconstr Surg 2015;136:897–912.
    1. Tremolada C, Palmieri G, Ricordi C. Adipocyte transplantation and stem cells: Plastic surgery meets regenerative medicine. Cell Transplant 2010;19:1217–1223.
    1. Zuk PA, Zhu M, Mizuno H et al. Multilineage cells from human adipose tissue: Implications for cell‐based therapies. Tissue Eng 2001;7:211–228.
    1. Schäffler A, Büchler C. Concise review: Adipose tissue‐derived stromal cells—Basic and clinical implications for novel cell‐based therapies. Stem Cells 2007;25:818–827.
    1. Yañez R, Lamana ML, García‐Castro J et al. Adipose tissue‐derived mesenchymal stem cells have in vivo immunosuppressive properties applicable for the control of the graft‐versus‐host disease. Stem Cells 2006;24:2582–2591.
    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;8:315–317.
    1. Gronthos S, Mankani M, Brahim J et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–13630.
    1. Lama VN, Smith L, Badri L et al. Evidence for tissue‐resident mesenchymal stem cells in human adult lung from studies of transplanted allografts. J Clin Invest 2007;117:989–996.
    1. Zheng B, Cao B, Crisan M et al. Prospective identification of myogenic endothelial cells in human skeletal muscle. Nat Biotechnol 2007;25:1025–1034.
    1. Gargett CE, Schwab KE, Zillwood RM et al. Isolation and culture of epithelial progenitors and mesenchymal stem cells from human endometrium. Biol Reprod 2009;80:1136–1145.
    1. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res 2003;18:694–704.
    1. Schwab KE, Gargett CE. Co‐expression of two perivascular cell markers isolates mesenchymal stem‐like cells from human endometrium. Hum Reprod 2007;22:2903–2911.
    1. Crisan M, Yap S, Casteilla L et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell 2008;3:301–313.
    1. da Silva ML, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells 2008;26:2287–2299.
    1. Traktuev DO, Merfeld‐Clauss S, Li J et al. A population of multipotent CD34‐positive adipose stromal cells share pericyte and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothelial networks. Circ Res 2008;102:77–85.
    1. Stefanska A, Kenyon C, Christian HC et al. Human kidney pericytes produce renin. Kidney Int 2016;90:1251–1261.
    1. Guimarães‐Camboa N, Cattaneo P, Sun Y et al. Pericytes of multiple organs do not behave as mesenchymal stem cells in vivo. Cell Stem Cell 2017;20:345–359.
    1. Corselli M, Chen CW, Sun B et al. The tunica adventitia of human arteries and veins as a source of mesenchymal stem cells. Stem Cells Dev 2012;21:1299–1308.
    1. de Souza LEB, de Malta TM, Kashima Haddad S et al. Mesenchymal stem cells and pericytes: To what extent are they related? Stem Cells Dev 2016;25:1843–1852.
    1. James AW, Zara JN, Zhang X et al. Perivascular stem cells: A prospectively purified mesenchymal stem cell population for bone tissue engineering. Stem Cells Translational Medicine 2012;1:510–519.
    1. Kramann R, Goettsch C, Wongboonsin J et al. Adventitial MSC‐like cells are progenitors of vascular smooth muscle cells and drive vascular calcification in chronic kidney disease. Cell Stem Cell 2016;19:628–642.
    1. Hindle P, Khan N, Biant L et al. The infra‐patellar fat pad as a source of perivascular stem cells with increased chondrogenic potential for regenerative medicine. Stem Cells Translational Medicine 2017;6:77–87.
    1. Crisan M, Corselli M, Chen WCW et al. Perivascular cells for regenerative medicine. J Cell Mol Med 2012;16:2851–2860.
    1. Corselli M, Chin C, Parekh C et al. Perivascular support of human hematopoietic stem/progenitor cells. Blood 2013;121:2891–2901.
    1. Zimmerlin L, Donnenberg VS, Rubin JP et al. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry 2013;83:134–140.
    1. Vezzani B, Pierantozzi E, Sorrentino V. Not all pericytes are born equal: pericytes from human adult tissues present different differentiation properties. Stem Cells Dev 2016;25:1549–1558.
    1. Vezzani B, Pierantozzi E, Sorrentino V. Mesenchymal stem cells: From the perivascular environment to clinical applications. Histol Histopathol 2018;7:11998.
    1. da Silva ML, Maistro Malta T, Panepucci RA et al. Transcriptomic comparisons between cultured human adipose tissue‐derived pericytes and mesenchymal stromal cells. Genom Data 2016;7:20–25.
    1. Hardy WR, Moldovan NI, Moldovan L et al. Transcriptional networks in single perivascular cells sorted from human adipose tissue reveal a hierarchy of mesenchymal stem cells. Stem Cells 2017;35:1273–1289.
    1. Tang W, Zeve D, Suh JM et al. White fat progenitor cells reside in the adipose vasculature. Science 2008;322:583–586.
    1. Feng J, Mantesso A, De Bari C et al. Dual origin of mesenchymal stem cells contributing to organ growth and repair. Proc Natl Acad Sci U S A 2011;108:6503–6508.
    1. Zhao H, Feng J, Seidel K et al. Secretion of shh by a neurovascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor. Cell Stem Cell 2014;14:160–173.
    1. Dellavalle A, Maroli G, Covarello D et al. Pericytes resident in postnatal skeletal muscle differentiate into muscle fibres and generate satellite cells. Nat Commun 2011;2:499.
    1. Krautler NJ, Kana V, Kranich J et al. Follicular dendritic cells emerge from ubiquitous perivascular precursors. Cell 2012;150:194–206.
    1. Göritz C, Dias DO, Tomilin N et al. A pericyte origin of spinal cord scar tissue. Science 2011;333:238–242.
    1. Dulauroy S, Di Carlo SE, Langa F et al. Lineage tracing and genetic ablation of ADAM12(+) perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat Med 2012;18:1262–1270.
    1. Kramann R, Schneider RK, DiRocco DP et al. Perivascular Gli1+ progenitors are key contributors to injury‐induced organ fibrosis. Cell Stem Cell 2015;16:51–66.
    1. Murray IR, Gonzalez ZN, Baily J et al. αv integrins on mesenchymal cells critically regulate skeletal and cardiac muscle fibrosis. Nat Commun 2017;8:1118.
    1. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000;113:1161–1166.
    1. Guilak F, Lott KE, Awad HA et al. Clonal analysis of the differentiation potential of human adipose‐derived adult stem cells. J Cell Physiol 2006;206:229–237.
    1. Russell KC, Phinney DG, Lacey MR et al. In vitro high‐capacity assay to quantify the clonal heterogeneity in trilineage potential of mesenchymal stem cells reveals a complex hierarchy of lineage commitment. Stem Cells 2010;28:788–798.
    1. Manini I, Gulino L, Gava B et al. Multi‐potent progenitors in freshly isolated and cultured human mesenchymal stem cells: A comparison between adipose and dermal tissue. Cell Tissue Res 2011;344:85–95.
    1. Tabit CJ, Slack GC, Fan K et al. Fat grafting versus adipose‐derived stem cell therapy: Distinguishing indications, techniques, and outcomes. Aesthetic Plast Surg 2012;36:704–713.
    1. Bianchi F, Maioli M, Leonardi E et al. A new non enzymatic method and device to obtain a fat tissue derivative highly enriched in pericyte‐like elements by mild mechanical forces from human lipoaspirates. Cell Transplant 2013;2:2063–2077.
    1. Raffaini M, Tremolada C. Micro fractured and purified adipose tissue graft (Lipogems®) can improve the orthognathic surgery outcomes both aesthetically and in postoperative healing. CellR4 2014;2:e1118.
    1. Cestaro G, De Rosa M, Massa S et al. Intersphincteric anal lipofilling with micro‐fragmented fat tissue for the treatment of faecal incontinence: preliminary results of three patients. Wideochir Inne Tech Maloinwazyjne 2015;10:337–341.
    1. Fantasia J, Chen H, Santos Cortes JA. Microfractured and purified adipose tissue (Lipogems™ system) injections for treatment of atrophic vaginitis. J Urol Res 2016;3:1073–1075.
    1. Saibene AM, Pipolo C, Lorusso R et al. Transnasal endoscopic microfractured fat injection in glottic insufficiency. B‐ENT 2015;11:229–234.
    1. Giori A, Tremolada C, Vailati R et al. Recovery of function in anal incontinence after micro‐fragmented fat graft (Lipogems®) injection: Two years follow up of the first 5 cases. CellR4 2015;3:e1544.
    1. Tremolada C, Beltrami G, Magri A et al. Adipose mesenchymal stem cells and regenerative adipose tissue graft (Lipogems®) for musculoskeletal regeneration. Eur J Muscoloskeletal Dis 2014;3:57–67.
    1. Striano RD, Chen H, Bilbool N et al. Non‐responsive knee pain with osteoarthritis and concurrent meniscal disease treated with autologous micro‐fragmented adipose tissue under continuous ultrasound guidance. CellR4 2015;3:e1690.
    1. Randelli P, Menon A, Ragone V et al. Lipogems product treatment increases the proliferation rate of human tendon stem cells without affecting their stemness and differentiation capability. Stem Cells Int 2016;2016:4373410.
    1. Bianchi F, Olivi E, Baldassarre M et al. Lipogems®, a new modality off at tissue handling to enhance tissue repair in chronic hind limb ischemia. CellR4 2014;2:e1289.
    1. Benzi R, Marfia G, Bosetti M et al. Microfractured lipoaspirate may help oral bone and soft tissue regeneration: A case report. CellR4 2015;3:e1583.
    1. Schindelin J, Arganda‐Carreras I, Frise E et al. Fiji: An open‐source platform for biological image analysis. Nat Methods 2012;9:676–682.
    1. West CC, Hardy WR, Murray IR et al. Prospective isolation of perivascular stem cells (PSC) from human adipose tissue: Cell population metrics across a large cohort of diverse demographics. Stem Cell Res Ther 2016;7:47.
    1. Caplan AI. Mesenchymal stem cells: Time to change the name! Stem Cells Translational Medicine 2017;6:1445–1451.
    1. Phinney DG, Pittenger MF. Concise review: MSC‐derived exosomes for cell‐free therapy. Stem Cells 2017;35:851–858.
    1. Zimmerlin L, Donnenberg VS, Pfeifer ME et al. Stromal vascular progenitors in adult human adipose tissue. Cytometry A 2010;77:22–30.
    1. Ceserani V, Ferri A, Berenzi A et al. Angiogenic and anti‐inflammatory properties of micro‐fragmented fat tissue and its derived mesenchymal stromal cells. Vasc Cell 2016;18:8–3.
    1. Mendel TA, Clabough EBD, Kao DS et al. Pericytes derived from adipose‐derived stem cells protect against retinal vasculopathy. PLoS One 2013;8:e691.
    1. König MA, Canepa DD, Cadosch D et al. Direct transplantation of native pericytes from adipose tissue: A new perspective to stimulate healing in critical size bone defects. Cytotherapy 2016;18:41–52.
    1. West CC, Hardy WR, Murray IR et al. Prospective purification of perivascular presumptive mesenchymal stem cells from human adipose tissue: Process optimization and cell population metrics across a large cohort of diverse demographics. Stem Cell Res Ther 2016;7:47.
    1. Zhang J, Du C, Guo W et al. Adipose tissue‐derived pericytes for cartilage tissue engineering. Curr Stem Cell Res Ther 2017;12:513–521.
    1. Nigro E, Scudiero O, Monaco ML et al. New insight into adiponectin role in obesity and obesity‐related diseases. Biomed Res Int 2014;2014:658913.
    1. Ronti T, Lupattelli G, Mannarino E. The endocrine function of adipose tissue: An update. Clin Endocrinol (Oxf) 2006;64:355–365.
    1. Yang Q, Graham TE, Mody N et al. Serum retinol binding protein 4 contributes to insulin resistance in obesity and type 2 diabetes. Nature 2005;436:356–362.
    1. Arend WP. The balance between IL‐1 and IL‐1Ra in disease. Cytokine Growth Factor Rev 2003;13:323–340.
    1. Fasshauer M, Blüher M. Adipokines in health and disease. Trends Pharmacol Sci 2015;36:461–470.
    1. Sheng J, Xu Z. Three decades of research on angiogenin: A review and perspective. Acta Biochim Biophys Sin 2016;48:399–410.
    1. Nassiri F, Cusimano MD, Scheithauer BW et al. Endoglin (CD105): A review of its role in angiogenesis and tumor diagnosis, progression and therapy. Anticancer Res 2011;31:2283–2290.
    1. De Falco S, Gigante B, Persico GM. Structure and function of placental growth factor. Trends Cardiovasc Med 2002;12:241–246.
    1. Bussolino F, Di Renzo MF, Ziche M et al. Hepatocyte growth factor is a potent angiogenic factor which stimulates endothelial cell motility and growth. J Cell Biol 1992;119:629–641.
    1. Liu ZJ, Battinelli E, Sparger KA et al. Novel insights into the role of platelet angiogenic growth factors on the regulation of normal vascular development. Paper presented at: ASH 59th Annual Meeting and Exposition, Atalanta, 2017.
    1. Bruns AF, Smith J, Yuldasheva N et al. Insulin‐like growth factor binding protein 2 (igfbp2): A positive regulator of angiogenesis? BMJ Heart 2017;103:A121.
    1. Bodnar RJ. Chemokine regulation of angiogenesis during wound healing. Adv Wound Care 2015;4:641–650.
    1. Yates‐Binder CC, Rodgers M, Jaynes J et al. An IP‐10 (CXCL10)‐derived peptide inhibits angiogenesis. PLoS One 2012;7:e40812.
    1. Cao Y, Ji RW, Davidson D et al. Kringle domains of human angiostatin. Characterization of the anti‐proliferative activity on endothelial cells. J Biol Chem 1996;271:29461–29467.
    1. Volpert OV, Tolsma SS, Pellerin S et al. Inhibition of angiogenesis by thrombospondin‐2. Biochem Biophys Res Commun 1995;217:326–332.
    1. O’Rahilly S. GDF15—From biomarker to allostatic hormone. Cell Metab 2017;26:807–808.
    1. Calandra T, Roger T. Macrophage migration inhibitor factor: A regulator of innate immunity. Nat Rev Immunol 2003;3:791–800.
    1. Müller M, Carter S, Hofer MJ et al. The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 in neuroimmunity—A tale of conflict and conundrum. Neuropathol Appl Neurobiol 2010;36:368–387.
    1. Comerford I, Harata‐Lee Y, Bunting MD et al. A myriad of functions and complex regulation of the CCR7/CCL19/CCL21 chemokine axis in the adaptive immune system. Cytokine Growth Factor Rev 2013;24:269–283.
    1. Appay V, Rowland‐Jones SL. RANTES: A versatile and controversial chemokine. Trends Immunol 2001;22:83–87.
    1. Schonbeck U, Libby P. The CD40/CD154 receptor/ligand dyad. Cell Mol Life Sci 2001;58:4–43.
    1. Crewe C, An YA, Scherer PE. The ominous triad of adipose tissue dysfunction: Inflammation, fibrosis, and impaired angiogenesis. J Clin Invest 2017;127:74–82.
    1. Suffee N, Richard B, Hlawaty H et al. Angiogenic properties of the chemokine RANTES/CCL5. Biochem Soc Trans 2011;39:1649–1653.
    1. Francescone R, Ngernyuang N, Yan W et al. Tumor‐derived mural‐like cells coordinate with endothelial cells: role of YKL‐40 in mural cell‐mediated angiogenesis. Oncogene 2014;33:2110–2122.
    1. Murray IR, Peault B. Q&A: What is a mesenchymal stem cell, and why is it important? BMC Biol 2015;13:99.
    1. Van den Brink SC, Sage F, Vértesy Á et al. Single‐cell sequencing reveals dissociation‐induced gene expression in tissue subpopulations. Nat Methods 2017;14:935–936.
    1. García‐Contreras M, Messaggio F, Jimenez O et al. Differences in exosome content of human adipose tissue processed by non‐enzymatic and enzymatic methods. CellR4 2014;3:e1423.

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