Adipose-Derived Stem Cells for Regenerative Medicine

Lipomas and Liposarcomas

Various soft tissue tumors lend further weight to the existence of adipose-derived stem cells. Lipomas and liposarcomas are the most common diagnoses of soft tissue tumors presenting in a clinical setting. Often, these tumors occur in subcutaneous or visceral adipose depots and proliferate slowly. The lipid content of benign lipomas and well differentiated liposarcomas is comparable to that seen in normal adipose tissue. This contrasts to myxoid and dedifferentiated liposarcomas, which contain less triglyceride.¹³ Using noninvasive nuclear magnetic resonance detection, these distinctions can be used for diagnostic purposes. The quintessential nuclear hormone receptor associated with adipogenesis, peroxisome proliferator-activated receptor (PPAR)γ, can be found in all histological grades of liposarcomas. Ligands for the PPARγ transcription factor include natural (long-chain fatty acids, prostaglandin J2) and synthetic (thiazolidinedione) compounds,¹⁴ and, in vitro, these agents can induce liposarcoma-derived cells to differentiate into adipocytes.¹⁵ Based on these observations, physicians have used oral thiazolidinediones to treat patients with liposarcomas, thereby reducing cell proliferation and increasing expression of adipocyte gene markers.^16,17 Although it remains to be determined whether ligand-induced adipogenesis is an effective chemotherapy for patients with liposarcoma, the work is consistent with the hypothesis that liposarcomas derive from a stem cell progenitor.

Cell Isolation and Mechanical Devices

The initial methods to isolate cells from adipose tissue were pioneered by Rodbell^32,33 and Rodbell and Jones³⁴ in the 1960s. They minced rat fat pads, washed extensively to remove contaminating hematopoietic cells, incubated the tissue fragments with collagenase, and centrifuged the digest, thereby separating the floating population of mature adipocytes from the pelleted stromal vascular fraction (SVF) (Figure). The SVF consisted of a heterogeneous cell population, including circulating blood cells, fibroblasts, pericytes, and endothelial cells as well as “preadipocytes” or adipocyte progenitors.^32–34 The final isolation step selected for the plastic adherent population within the SVF cells, which enriched for the “preadipocytes.” Subsequently, this procedure has been modified for the isolation of cells from human adipose tissue specimens.^35–39 Initially, fragments of human tissue were minced by hand; however, with the development of liposuction surgery, this procedure has been simplified. During tumescent liposuction, plastic surgeons infuse the subcutaneous tissues with a saline solution containing anesthetic and/or epinephrine via a cannula and then remove both the liquid and tissue under suction.⁴⁰ The procedure generates finely minced tissue fragments, the size of which depends on the dimensions of the cannula. Independent studies have determined that liposuction aspiration alone does not significantly alter the viability of isolated SVF cells.^41–43 Indeed, adherent stromal cells with characteristics of adipocyte progenitors can be found directly within the liposuction aspiration fluid, as well as in SVF derived from the tissue fragment digests.⁴⁴ However, when ultrasound-assisted liposuction is performed, the number of cells recovered from tissue digests is reduced, as is their proliferative capacity.⁴³ The recovery of ASCs can be improved further by manipulating the centrifugation speed.⁴⁵ Investigators have achieved optimal cell recovery using a centrifugation speed of 1200g based on the subsequent formation of a human-derived adipose tissue depot following implantation in an immunodeficient murine model.⁴⁵

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Figure

Processing of lipoaspirate and isolation of adipose-derived stem cells.

The cell isolation process requires the manipulation of large volumes of lipid-laden cells, presenting potential risks to equipment and personnel. To facilitate the process, several groups have fabricated devices to automate the cell isolation.^4,46 One approach uses a “bag within a bag.” The suctioned aspirate flows through a central bag that automatically sieves the tissue while draining away the aspiration fluid. Subsequently, the trapped tissue can be washed and further manipulated.⁴ Others have developed a closed, rotating, controlled temperature incubator capable of collagenase digesting and separating up to one liter of tissue at a time.⁴⁶ These prototypes may one day lead to commercially available manufactured devices for large scale, automated adipose tissue manipulation and cell isolation suitable for clinical applications.

Immunophenotype

Multiple independent groups have examined the surface immunophenotype of ASCs isolated from human and other species (Table 1).^{44,47–51–58} The expression profile changes as a function of time in passage and plastic adherence.^56,57 After 2 or more successive passages in culture, the ASCs express characteristic adhesion and receptor molecules, surface enzymes, extracellular matrix and cytoskeletal proteins, and proteins associated with the stromal cell phenotype. Despite any differences in the isolation and culture procedures, the immunophenotype is relatively consistent between laboratories (Table 1). Indeed, the surface immunophenotype of ASCs resembles that of bone marrow–derived mesenchymal stem or stromal cells (MSCs)⁵⁹ and skeletal muscle-derived cells.⁶⁰ Direct comparisons between human ASC and MSC immunophenotypes are >90% identical.⁵² Consistent with this, the 2 cell populations display similar mitogen-activated protein kinase phosphorylation in response to tumor necrosis factor-a, lipolytic responses to β-adrenergic agents, and adiponectin and leptin secretion following adipogenesis.^61,62 Nevertheless, differences in surface protein expression have been noted between ASCs and MSCs. For example, the glycoprotein CD34 is present on human ASCs early in passage but has not been found on MSCs.^57,59 Not all differences reported in the literature for specific markers are necessarily valid. The Stro-1 antigen, a classic bone marrow MSC-associated surface antigen,⁶³ was reported as both absent⁵⁰ and present⁵² on human ASCs. Differences in Stro-1 antibody sources and detection methods (flow cytometry versus immunohistochemistry) could account for this apparent discrepancy in findings.

Identification of the ASC surface immunophenotype has provided a mechanism to enrich or purify the stem cell population directly from the heterogeneous SVF cells.^64–66 Investigators have used immunomagnetic beads or flow cytometry to both positively and negatively select for a subpopulation of cells within the SVF. For example, endothelial progenitors can be removed by negatively selecting for cells expressing CD31 or platelet endothelial cell adhesion molecule-1.^58,64–67 Likewise, positive selection has been performed using CD34 and other antigens. One group working with human adipose tissue has demonstrated that the CD34 positive SVF cell population contained the ASCs.^65,66 Species variations may exist; however, studies of murine adipose tissue found that the CD34-negative SVF population was enriched for ASCs based on an in vitro differentiation and in vivo ischemia reperfusion assays.^58,68

Transcriptome and Proteome

Gene microarrays studies analyzing the transcriptomes of undifferentiated human ASCs and bone marrow–derived MSCs have been reported by independent laboratories.^55,69,70 An analysis of a panel of 28 genes was not significantly different between the 2 cell types.⁶⁹ A more comprehensive comparison using Affymetrix gene chips has determined that human ASCs and MSCs share a common transcriptome⁷¹ (R. Izadpanah, B. Bunnell, C. Kriedt, unpublished observation, 2006). The correlation coefficient between the transcriptomes of ASCs and MSCs derived from multiple donors was approximately 50%; this compares to an average correlation coefficient of 71% and 64% between individual donors within the ASC and MSC groups, respectively.⁷⁰ The ASCs and MSCs derived from both human and rhesus monkeys express stem cell–associated gene markers, including Oct4, Rex1, and Sox2.⁷² Overall, these data suggest that ASC and MSC display distinct but similar expression profiles based on mRNA analyses.

Analyses of the ASC and adipocyte proteome by mass spectrometry and other approaches have documented the identity of >200 proteins in both the undifferentiated and adipose differentiated cells.^73–79 The human ASC proteome shares features in common to that reported for fibroblasts, MSCs, and other lineages.^74,80,81 A direct comparison of ASC and MSC proteomes based on 2D gel electrophoresis has identified 19 individual proteins with >1.5-fold differences (R. Izadpanah, B. Bunnell, C. Kriedt, I. Kheterpal, A. White, unpublished observation, 2006).

Cell Proliferation and Culture

In vitro, ASCs display a cell doubling time of 2 to 4 days, depending on the culture medium and passage number.^56,72 The ASCs maintain their telomere length with progressive passage in culture^72,82; however, reports differ as to whether the telomerase activity of the ASCs is maintained,⁸² decreased with progressive passage,⁷² or absent.⁵⁵ Interspecies variation does occur; the characteristics of human and rhesus ASCs are similar but not identical.⁷² With prolonged passage for >4 months, human ASCs have been observed to undergo malignant transformation.⁸³ In at least one laboratory, serially passaged ASCs displayed karyotypic abnormalities at a frequency of >30% and, when implanted into immunodeficient mice, formed tumors at a frequency of 50%.⁸³ These findings indicate that caution should be exercised in the manipulation and culture of the adipose-derived cells. As is discussed below, safety testing for any clinical ASC products will need to include karyotype analysis as well as in vitro and in vivo tumorigenesis assays.

Mechanisms of Potential Utility: How Do ASCs work?

Investigators have postulated a number of nonexclusive mechanisms through which ASCs can be used to repair and regenerate tissues. First, ASCs delivered into an injured or diseased tissue may secrete cytokines and growth factors that stimulate recovery in a paracrine manner. The ASCs would modulate the “stem cell niche” of the host by stimulating the recruitment of endogenous stem cells to the site and promoting their differentiation along the required lineage pathway. In a related manner, ASCs might provide antioxidants chemicals, free radical scavengers, and chaperone/heat shock proteins at an ischemic site. As a result, toxic substances released into the local environment would be removed, thereby promoting recovery of the surviving cells. Exciting studies have suggested that transplanted bone marrow– derived MSCs can deliver new mitochondria to damaged cells, thereby rescuing aerobic metabolism.⁸⁴ It may develop that similar studies in ASCs will uncover a comparable ability to contribute mitochondria. A final mechanism is to differentiate ASCs along a desired lineage, as described in the following sections and Table 2.

In all contexts, it is important to consider the potential use of both autologous and allogeneic ASCs. Autologous ASCs offer advantages from regulatory, histocompatibility, and infectious perspectives. As seen with blood cell products, it is often not feasible for an individual patient to provide his or her own therapeutic cell product. Recent studies support further evaluation of allogeneic ASC transplantation. Independent studies from 3 laboratories have determined that passaged human ASCs, as opposed to freshly isolated SVF cells, reduce their expression of surface histocompatibility antigens and no longer stimulate a mixed lymphocyte reaction when cocultured with allogeneic peripheral blood monocytes.^57,85,86 Like bone marrow–derived MSCs,⁸⁷ the ASCs actually suppress immunoreactions.^57,85 This indicates that the ASCs may not elicit a cytotoxic T-cell response in vivo. The hypothesis that transplanted allogeneic ASCs will not elicit a robust immune response and subsequent rejection needs independent and comprehensive testing. If correct, such a finding would have a profound impact on the application of ASCs in regenerative medicine. The ability to transplant allogeneic ASCs will reduce the cost of cell therapies. Quality assurance and control steps can be stream-lined once multiple use, as opposed to single donor/recipient, product approval takes place and ASCs are manufactured in large volumes. Likewise, the availability of off-the-shelf allogeneic ASCs will allow physicians and surgeons to use them directly at the point of care, i.e., the emergency room, rather than limiting their use to elective procedures.

Endothelial

The preadipocytes within adipose tissue depots and endothelial cells exhibit close relationships in vivo.⁹⁷ Following isolation, cells within the heterogeneous SVF express markers consistent with an endothelial phenotype including CD31, CD144 (VE-cadherin), and von Willebrand factor.^{65,68,98–100} These should be distinguished from ASCs because they have demonstrated lineage commitment. Williams and colleagues pioneered the isolation of such differentiated endothelial cells from human adipose tissue.^48,101 By screening for collagenase enzymes with specific characteristics, they recovered cells expressing von Willebrand factor.¹⁰² They were able to seed these endothelial cells onto synthetic vascular grafts and improve the patency of these grafts following surgery.^47,48,101 Alternatively, they successfully transplanted the cells into the corpus cavernosum of rats, suggesting that they could be used in the treatment of erectile dysfunction.¹⁰³

Others have documented the ability of SVF cells and culture expanded ASCs to undergo endothelial differentiation in vitro.^{65,66,68,98–100,104} Planat-Benard et al⁹⁸ observed that CD13⁺CD34⁺ SVF cells cultured in Matrigel expressed CD31 and von Willebrand factor. Moreover, the cells formed branching networks, consistent with the formation of vascular structures. Miranville et al⁶⁵ reported similar findings using a population of CD31⁻CD34⁺ SVF cells. The addition of VEGF enhanced their differentiation, based on expression of CD31 and the development of vascular structures in Matrigel. In related work, Cao et al⁶⁸ demonstrated that a population of CD31⁻CD34⁻CD106⁻, fetal liver kinase-1⁺ (flk-1) adipose-derived cells could express endothelial markers in response to VEGF. Likewise, Martinez-Estrada et al¹⁰⁴ found that flk-1⁺ cells isolated from cultured ASCs displayed an endothelial phenotype in the presence of VEGF. In vivo studies further support the endothelial differentiation potential of SVF cells. In a rat ischemic hindlimb model, the introduction of the CD31⁻ population improves angiogenesis and subsequent recovery of vascular supply.^{65,68,98,99,105} This finding has been confirmed by at least 5 independent groups and is reproduced both when the cells are delivered intravenously^65,68,99 or by intramuscular injection in the direct vicinity of the ischemic injury.^98,105 Although the ASCs can integrate as fully functional and differentiated endothelial cells in vivo, they may contribute additionally through paracrine pathways. The adipose-derived cells secrete angiogenic cytokines such as VEGF and hepatocyte growth factor (HGF), and these are postulated to contribute to the ASC angiogenic properties.^99,105 The levels of VEGF and/or HGF secreted by ASCs can be induced by exposure of the cells to hypoxia,⁹⁹ growth and differentiation factor 5,¹⁰⁶ tumor necrosis factor-α,¹⁰⁷ basic fibroblast growth factor, epidermal growth factor, and ascorbate.¹⁰⁸ In vivo studies using the murine 3T3-F442A preadipocyte model suggest that a reciprocal relationship may exist between angiogenesis and adipogenesis.¹⁰⁹ This involves VEGF, because the introduction of VEGF receptor antibodies interferes with the formation of new adipose depots in vivo, which is consistent with a paracrine pathway between endothelial and preadipocyte cells.¹⁰⁹ Further support for a paracrine relationship between these lineages comes from the observation of increased proliferative rates noted in 2D and 3D cocultures of preadipocytes and endothelial cells.^64,110

Smooth Muscle

In vitro, human ASCs can express a-smooth muscle actin, calponin, and SM22, consistent with a smooth muscle phenotype.^111,112 The addition of transforming growth factor-β and sphingosylphosphorylcholine induces human ASC expression of smooth muscle–associated markers.^111,112 Over-expression studies in a preadipocyte murine model indicate that the aortic carboxypeptidase-like protein may underlie the mechanism of ASC smooth muscle differentiation.^113,114 In addition, mechanical stimuli modulate the smooth muscle phenotype; cyclic uniaxial strain reduced human ASC expression of α-smooth muscle actin and calponin.¹¹⁵ In vivo, human ASCs persist for up to 12 weeks and display the morphology of smooth muscle cells when injected into the urinary tract of immunodeficient mice.¹¹⁶ Technological developments allowing for the controlled generation and manipulation of ASC sheets may provide methods to deliver smooth muscle– differentiated ASCs for genitourinary and cardiovascular regenerative procedures.^95,117

Evidence for Additional Lineage Differentiation

There is a growing body of experimental evidence from both in vitro and in vivo studies demonstrating the multi-potentiality of ASCs from adipose tissue isolated from humans and other species. These include the adipocyte,^52,118–120 chondrocyte,^52,120–122 hematopoietic supporting,^108,123 hepatocyte,^124,125,126 neuronal-like,^{52,53,127–130} osteoblast,^{52,120,131–133} pancreatic,¹³⁴ and skeletal myocyte^{52,120,135,136} pathways. Because these lineage pathways fall outside of the scope of the current review, we refer the reader to in-depth reviews for further information on these subjects.^88,137–139

Moving Into the Future: The Challenges

Many important scientific and medical questions remain. These include the development of large scale manufacturing methods with appropriate quality assurance and quality control to generate cells in compliance with current Good Manufacturing Practices. Preferably, these steps will be performed in a closed, sterile container that minimizes risk for contamination of the tissue while protecting the personnel from exposure to blood-borne pathogens. Cell culture in stirred or rotating flasks, perfused hollow fiber bioreactors, or Teflon bags may be required. It will be valuable to develop a serum protein free culture medium to avoid issues related to bovine spongiform encephalopathy or other xenogeneic infections. Finally, it will be important to determine how to store the ASCs, what kind of containers to store them in, how to label those containers, how to ship them to the point of care, and how long their shelf life will be.

Recent studies have demonstrated that human ASCs, after prolonged culture in vitro, are capable of forming tumors in immunodeficient mice.⁸³ Consequently, long-term experiments examining the safety of ASC transplantation in appropriate animal models will be required. Such studies should be designed in consultation with regulatory agencies to insure that all preclinical questions are addressed before advancing to phase I studies in patients. If and when ASCs are approved for clinical use, physicians and health care professionals will need to be educated regarding their unique properties and correct application.

We thank Drs A. Bouloumie, L. Casteilla, J. Jung, K. Marra, J. P. Rubin, C. Sengenes, and K. Yoshimura for providing articles and discussion during the preparation of this review; Drs B. Kozak and R. Martin for critical reading of the manuscript; and P. Browne, T. Nguyen, X. Wu, and G. Yu for assistance in preparation of the figure.

Sources of Funding

This work was supported in part by funding from the Pennington Biomedical Research Center Clinical Nutrition Research Unit (P30 DK072476) and the Pennington Biomedical Research Foundation. The research at the Tulane University Primate Research Center was supported by the National Center for Research Resources, NIH grant RR00164, and a grant from the State of Louisiana Millennium Health Excellence Fund and the Louisiana Gene Therapy Research Consortium.