Adipose Derived Cells and Tissues for Regenerative Medicine - ACS

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Adipose Derived Cells and Tissues for Regenerative Medicine Jeffrey M. Gimble, Stephen P. Ray, Fabiana Zanata, James Wade, Kamran Khoobehi, Xiying Wu, Lydia Masako Ferreira, and Bruce A. Bunnell ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00261 • Publication Date (Web): 24 Jun 2016 Downloaded from http://pubs.acs.org on June 26, 2016

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ACS Biomaterials Science & Engineering

Journal:

ACS Biomaterials Science & Engineering™

Editor in Chief:

David Kaplan PhD

Special Issue Guest Editor:

Kaiming Ye PhD

Title:

Adipose Derived Cells and Tissues for Regenerative Medicine

Authors:

Jeffrey M. Gimble1-3, Stephen P. Ray4, Fabiana Zanata1,5, Lydia Masako Feirrera5, James Wade6, Kamran Khoobehi7, Xiying Wu3, Bruce A. Bunnell1,8,9

Affiliations: 1Center for Stem Cell Research & Regenerative Medicine and 2Departments of Medicine, Structural and Cellular Biology, and Surgery, Tulane University School of Medicine, and 3LaCell LLC, New Orleans LA USA 70112; 4Midwestern Regional Medical Center, Cancer Treatment Center of America, Zion IL USA 60099; 5Universidade Federal São Paulo, São Paulo, Brazil; Plastic Surgery Associates, Baton Rouge, LA USA 70808; Khoobehi and Associates, Metairie, LA, 70002; 8Department of Pharmacology, Tulane University School of Medicine, New Orleans LA USA 70112; and 9Tulane National Primate Research Center, Covington LA USA.

Abstract: Adipose tissue is now recognized as a complex organ serving endocrine, immune, and metabolic functions. Adipose depots are composed of mature adipocytes as well as stromal vascular fraction (SVF) cells, a heterogeneous population of B and T lymphocytes, endothelial cells, macrophages, pericytes, smooth muscle, and stromal cells that can be isolated by enzymatic digestion. When placed into culture medium, a subset of the SVF cells can adhere to the plastic surface and expand in number. This latter population, known as “adipose-derived stromal/stem cells” (ASC), exhibits tri-lineage (adipo-, chondro-, osteo-) differentiation potential. There are currently over 180 clinical studies underway world-wide exploring the regenerative medical application of SVF cells and ASC in a range of medical conditions. Plastic surgeons have a particular interest in the use of autologous fat and SVF enhanced fat for cosmetic and reconstructive surgical procedures. Orthopedic and craniofacial surgeons have begun to use ASC to treat bone and musculoskeletal defects with success. Furthermore, studies are underway to exploit the immunomodulatory function of ASC to treat immune-mediated disorders such as Crohn’s disease. Indeed, it is postulated that adipose tissue and cells modulate tissue regeneration and inflammatory responses through their secretion of paracrine factors. Continued advances in this emerging field will require harmonization of international standards and guidelines defining the release criteria as well as the safety and efficacy of adipose-derived cells and tissues. Currently there are no accepted standard guidelines for autologous fat harvesting technique, processing or method of injection. Close collaboration between academia, industry, and regulatory authorities will be necessary to insure that adipose-derived products are affordable and quality controlled throughout the globe. Keywords: Adipose-derived Stromal/stem Cells, Cell Therapy, Lipoaspirate, Regenerative Medicine, Stromal Vascular Fraction Cells

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Introduction and History: Over the past quarter century, the lay public, medical, and scientific communities have gained a greater appreciation of adipose tissue depots in health and disease. While conventional thinking had confined adipose tissue exclusively to a subservient role in energy storage and metabolism, the identification in the early 1990’s of leptin as the first “adipokine” led to the recognition of adipose tissue as the body’s largest endocrine organ 1. Since then, multiple independent studies have shown that adipose tissue secretes additional adipokines, including adiponectin, resistin, serpins, and visfatin, among others 2. These factors act systemically to modulate metabolic function in central (brain) and peripheral (bone, heart, liver, skeletal muscle) organs. Studies in the early 2000’s determined that peripheral adipose depots contained a reservoir of hematopoietic cells that previously had been associated primarily with the bone marrow microenvironment 3, 4. Investigators determined that the stromal vascular fraction (SVF) cells isolated from collagenase-digested adipose tissue could substitute for bone marrow aspirates in studies with lethally irradiated mice 3. While irradiated mice died within a few weeks, transplantation of SVF cells rescued irradiated mice as well as bone marrow-derived hematopoietic progenitor cells 3. Nearly simultaneously, in 2001, the first studies characterizing what are now known as “adipose-derived stromal/stem cells” (ASC) were published 5. Similar to bone marrow-derived mesenchymal stem cells or multipotent stromal cells (BMSC), adherent, culture expanded ASC derived from SVF cells differentiated along the adipocyte, chondrocyte, and osteoblast lineage pathways and expressed a discrete set of surface antigens 5-7. Furthermore, both ASC and BMSC displayed potent immunomodulatory and immunosuppressor functionality based on in vitro and in vivo assays 8-10. Thus, adipose tissue itself as well as its isolated cells have endocrine, hematopoietic, immunomodulatory, and multipotent differentiation potential. This manuscript will consider the clinical translation of this information and how it is directing the future of regenerative medicine. Basic Science and Clinical Translation: Since 2001, the number of publications relating to “ASC” has increased exponentially, reaching nearly ~10,000 in number with ~200,000 citations. These studies have examined multiple ASC features, including the isolation, characterization, cytokine production, differentiation, immunomodulatory function, and metabolic characteristics as a function of donor demographics (body mass index, age, gender, ethnicity) and depot of origin (subcutaneous, visceral, other). These observations have established a foundation of knowledge that has since inspired investigators to apply adipose-derived cells and tissues to a wide range of regenerative medical conditions. The U.S. government website www.clinicaltrials.gov has registered >180 national and international clinical trials involving ASC in application to a wide range of pathologies (Table 1) 11, 12. The most straightforward clinical studies use ASC in the context of established adipose tissue function. These include cosmetic and reconstructive surgical applications for breast augmentation and post-lumpectomy/mastectomy repair as well as scar and facial atrophy regeneration. More complex studies have begun to use ASC successfully to repair craniofacial and musculoskeletal defects. Furthermore, adipose cells are being applied in the context of radiation therapy, an important component of many cancer treatments. Radiation therapy leads to changes in tissue characteristics, including thickened skin, decreased vessel diameter, and altered number disorganization of collagen bundles, all of which contribute to poor or irreversible wound healing status. Both animal models and human clinical applications of SVF have demonstrated progressive regeneration of damaged tissue with neovascularization and collagen reorganization following radiation injury 13-15. Additionally, studies are underway to exploit the immunomodulatory


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properties of ASC to improve outcomes in immune-mediated disorders such as Crohn’s disease. These represent only a subset of the ambitious ongoing studies world-wide. Adipose Depots and Heterogeneity at the Cellular, Functional, and Metabolic Levels Flow cytometric analyses have confirmed the heterogeneity of the SVF cell population which includes adipocytes, B lymphocytes, endothelial cells, macrophages, myeloid cells, pericytes, smooth muscle cells, and T lymphocytes, among others 8, 16-21. Although mature adipocytes are just one cellular constituent of the adipose depot, due to their size, they constitute a substantial, if not the major, percentage of the adipose depot volume. Adipose tissue is associated with three major adipocyte cell types: white, brown, and beige/brite (brown/white) 22. White adipose tissue (WAT) is the most frequently studied adipocyte cell type. The majority of WAT is derived from the embryonic mesoderm and its differentiation is primarily controlled by the master regulatory transcription factors CCAAT/Enhancer Binding Protein α (CEBPα) and Peroxisome Proliferator Activated Receptor γ2 (PPARγ2) 23, 24 . WAT adipocytes accumulate and store neutral lipids in vacuoles through a coordinated action of lipogenesis and lipolysis. Brown adipose tissue (BAT) differs substantially from WAT based on its ability to convert stored energy directly into heat. This occurs through the expression of uncoupling protein 1 (UCP1), a transmembrane proton channel that “short circuits” the energy storage mechanism within the mitochondria. The expression of UCP1 is regulated in part by the transcription factors PRDM16 and PGC1α 25. Studies of BAT adipocyte transcriptome have linked their development more closely to skeletal muscle than to WAT adipocytes. Like skeletal muscle progenitors, BAT adipocytes derive from cells expressing the Myf5 transcription factor 26. Beige adipocytes display features in common with both WAT and BAT 27. Beige cells have been noted to appear within WAT depots following exposure to inductive stimuli, including exposure to β3 adrenergic receptor and PPARγ2 agonists. In contrast to BAT adipocytes, beige cells do not express Myf5 but unlike WAT adipocytes, they do express UCP1 and its upstream regulators 22. Indeed, it may be an over-simplification to distinguish anatomical adipose depots as “white’, “beige” or “brown”; each of these lineages may be present within a single depot. Recent studies of adipose tissue in the human neck have determined that the cell types can change depending on the depth of the biopsy; superficial cells display features of white adipocytes, intermediate depth cells display a beige phenotype while the deepest adipocytes exhibit a transcriptomic phenotype consistent with brown adipocytes 28, 29. Adipose tissue is dispersed throughout the body and its abundance changes as a function of age, physiology, and nutritional status. Its locations in man include: Subcutaneous – This predominantly WAT depot is associated with energy storage, metabolism, and mechanical insulation. With increased nutrition, the depot accumulates in volume through a combination of cell hypertrophy and hyperplasia. Visceral (intra-abdominal) – This predominantly WAT depot is associated with energy storage, metabolism, and the sterile inflammatory response associated with obesity and its co-morbidities (hypertension, hyperlipidemia, inflammatory cytokine elevations). Unlike subcutaneous WAT, the SVF cells in visceral WAT contains mesothelial cells which may contribute to its unique function. Supraclavicular and cervical depots – The first studies demonstrating the persistence of BAT in adult human subjects used PET scanning to localize glucose uptake to these anatomical depots. This discovery



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has led to a renewed interest in human BAT physiology and its utility as a target for anti-obesity drug therapy 30-32. Perinephric and pericardial depots – While both depots contain WAT, they may also be a site of brown or beige cell formation and function. In newborn infants, thermogenesis within these depots could protect the adjacent vital organs. Bone marrow – While new born infants display little if any bone marrow adiposity, the percentage of marrow volume occupied by adipocytes increases with advancing age 33. By the time of peak bone mass in the mid-30’s, an individual’s femoral bone will contain >50% fat. In contrast to subcutaneous fat, severe starvation or anorexia is associated with increased, not decreased, marrow adiposity. A growing body of evidence identifies marrow adipocytes as either brown or beige in origin 34, 35. Mechanical (infrapatellar, palmar, plantar, and retro-orbital) - Mechanical fat depots are located in musculoskeletal junctions associated with repetitive compressive forces due to locomotion or orbital rotation. The retro-orbital fat pad is unique due to its cranial location 36. Unlike adipose tissue in the torso, head and neck adipose depots have a neural crest as opposed to mesodermal developmental origin and this may contribute to differences in their metabolic and differentiation function 37, 38. Recent transcriptomic analyses and in vivo tracking of genetically labeled cells indicate that the embryonic dermal origin of an adipose progenitor cell can determine its developmental fate 39. Since cells of mixed dermal origins can co-localize, this may account for the identification of white, beige, and brown adipocytes as “mini-organs” within a single adult adipose depot 39. Mammary - In contrast to all other organs, the mammary gland undergoes substantial developmental changes in post-natal life. In females, puberty is associated with expansion of the breast epithelial ducts and its parenchyma, which includes an abundance of WAT adipocytes. During pregnancy and lactation, the epithelial cells proliferate while the lipid in the mammary gland and peripheral fat depots is mobilized for milk secretion.

Isolation Methods General and plastic surgeons can excise adipose tissue through invasive surgeries such spanniculectomy or abdominoplasty however, plastic surgeons are more likely to take a less invasive approach known as dry or tumescent liposuction. This technique involves the insertion of a cannula subcutaneously and, in the case of the tumescent approach, is followed by local infiltration with a saline solution supplemented with epinephrine to reduce bleeding and lidocaine to reduce pain 40, 41. After a few minutes, the cannula is attached to a low pressure suction device or syringe and the swollen adipose tissue is removed to a sterile container. While this lipoaspirate is often discarded, doctors are increasingly using it as a source of tissue and/or cells for cosmetic and reconstructive purposes. Surgeons initially developed manual approaches to process and wash the lipoaspirate in the operating room. In its simplest approach, the cleaned was injected directly back into the patient for soft tissue volume reposition and/or augmentation. Alternatively, the tissue was digested with an enzyme (collagenase) to free the SVF cells from the extracellular matrix and allow for their isolation. The SVF cells could then be mixed with lipaspirate to perform “cell assisted lipotransfers” (CAL) to improve subsequent vascularization and engraftment 42, 43. The tissue manipulation and enzymatic digestion were first performed manually by


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technical staff in the operating theater or in a nearby laboratory certified to meet current Good Manufacturing Practices (cGMP) where the tissue could be manipulated in a laminar flow biological safety cabinet. While manual procedures can be performed with a high level of reproducibility through the strict use of standard operating procedures (SOPs) and manufacturing record documentation, they remain subject to operator error and protocol deviations. Consequently, a number of companies began to develop closed system mechanical devices to collect, wash, digest, centrifuge, and separate the cellular or tissue components from lipoaspirates (Table 2) 44-47. Some instruments compress the lipoaspirate tissue mechanically, creating a microfractured adipose tissue where the adipocyte volume is significantly reduced while the number of stromal/stem cells capable of secreting paracrine factors is enriched. Other devices incorporate an enzymatic digestion process to isolate the SVF cells directly; these require up to an extra hour of time to complete the procedure. All of these instruments are intended primarily for use at the “point of care”. A number have received CE approval for clinical use in the European Union and many are already used widely throughout Asia. At this point in time, the majority are still undergoing clinical trials for approval by the U.S. Food and Drug Administration (FDA). Product Criteria Any cell or tissue product intended for allogeneic use must meet standard sterility release criteria tests for bacterial, endotoxin, mycoplasma, and viral contamination. Beyond this series of fundamental tests, there remains a need to develop a standardized release criteria for manipulated adipose derived cell and tissue products. Ideally, such release criteria would be: 1. Rapid, reliable, and reproducible; 2. Economical; 3. Quantifiable based on established, industry accepted metrics derived from functionally relevant outcome assays; 4. Harmonized and standardized internationally. One step taken to address this goal was the development of a joint consensus statement by the International Society for Cellular Therapy (ISCT) and the International Federation of Adipose Therapeutics and Science (IFATS). Experts representing both societies developed a series of guidelines defining the characteristics of ASC and SVF cells (Table 3) 48. The SVF cells were defined as those cells isolated from adipose tissue by enzymatic digestion but which had not yet adhered to plastic or any other growth surface. In contrast, the ASC were defined as the plastic adherent population derived from the SVF cells in culture. Additional distinguishing characteristics included colony forming unit-fibroblast (CFU-F) formation as a surrogate for proliferation and frequency, tri-lineage (adipo-, chondro-, osteo-) differentiation, and immunophenotype based on flow cytometry. Recent studies suggest that the surface antigen CD34, long associated with hematopoietic progenitors and stem cells, may have value in predicting the functional utility of adipose tissue and cells, at least with respect to adipogenesis and soft tissue reconstruction 49. Furthermore, assays evaluating immunomodulatory and immunosuppressive functionality may improve the characterization of adipose-derived cell populations on a lot by lot basis 50, 51 .

Future Directions and Conclusions



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Regulatory authorities such as the FDA continue to monitor the rapid evolution of the regenerative medical landscape. During the past decade, the source of stem cells used in registered clinical trials has shifted away from an almost exclusive reliance on bone marrow to the use of adipose, amniotic, placental, and other tissues in nearly 50% of studies 52. Industry and academia must provide the regulators and the public with tangible assurances that adipose derived cell and tissue products meet expected levels of safety and efficacy reliably and reproducibly. This will require national and international studies to monitor long term post-treatment outcomes for both benefits (longer lifespan, reduced morbidity, improved quality of life, reduced overall cost of therapy) and risks (association with complications, cancer, autoimmune disorders). Furthermore, new therapies must demonstrate economic benefits for both the patient and society at large. The science and manufacturing of adipose cells and tissue products is still in its infancy. As the field matures and improvements are introduced, it is likely that large scale manufacturing and marketplace competition will reduce the costs required to deliver adipose-based therapies to the individual patient. In several decades, it is anticipated that adipose tissue will have the same impact and scope that we now take for granted with blood cell transfusion products. Disclosures JMG is the co-owner, co-founder, and Chief Scientific Officer of LaCell LLC, a for-profit biotechnology company focusing on the distribution of research products promoting the clinical translation of adipose derived cells and tissues. FZ received financial support from CAPES, Brazil (Process BEX 1524/15-1). References 1. Friedman J. 20 years of leptin: leptin at 20: an overview. The Journal of endocrinology. 2014;223:T1-8. 2. Trayhurn P and Wood IS. Adipokines: inflammation and the pleiotropic role of white adipose tissue. The British journal of nutrition. 2004;92:347-55. 3. Cousin B, Andre M, Arnaud E, Penicaud L and Casteilla L. Reconstitution of lethally irradiated mice by cells isolated from adipose tissue. Biochemical and biophysical research communications. 2003;301:1016-22. 4. Cousin B, Casteilla L, Laharrague P, Luche E, Lorsignol A, Cuminetti V and Paupert J. Immunometabolism and adipose tissue: The key role of hematopoietic stem cells. Biochimie. 2015. 5. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP and Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue engineering. 2001;7:211-28. 6. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P and Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Molecular biology of the cell. 2002;13:4279-95. 7. Gimble JM, Katz AJ and Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circulation research. 2007;100:1249-60. 8. McIntosh K, Zvonic S, Garrett S, Mitchell JB, Floyd ZE, Hammill L, Kloster A, Di Halvorsen Y, Ting JP, Storms RW, Goh B, Kilroy G, Wu X and Gimble JM. The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem cells. 2006;24:1246-53. 9. McIntosh KR, Frazier T, Rowan BG and Gimble JM. Evolution and future prospects of adiposederived immunomodulatory cell therapeutics. Expert review of clinical immunology. 2013;9:175-84. 10. Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C, Taureau C, Cousin B, Abbal M, Laharrague P, Penicaud L, Casteilla L and Blancher A. Immunomodulatory effect of human adipose


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Adipose Derived Cells and Tissues for Regenerative Medicine Jeffrey M. Gimble, Stephen P. Ray, Fabiana Zanata, Lydia Masako Feirrera, James Wade, Kamran Khoobehi, Xiying Wu, Bruce A. Bunnell

Liposuction

Tissue Processing

SVF Cell Isolation

ASC Expansion

Cryopreservation for Future Use


Autologous or Allogeneic Cell Therapy

Adipose Derived Cells and Tissues for Regenerative Medicine - ACS

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