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OPEN ACCESS ARTICLE
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TISSUE-SPECIFIC STEM CELLS |
aIndiana Center for Vascular Biology and Medicine,
bDepartment of Cellular and Integrative Physiology,
cKrannert Institute of Cardiology,
dDepartment of Medical and Molecular Genetics,
eDepartment of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA;
fR.L. Roudebush, VA Medical Center, Indianapolis, Indiana, USA
Key Words. Adipose-derived stem cells • RNA interference • Paracrine • Hepatocyte growth factor
Correspondence: Keith L. March, M.D., Ph.D., Indiana Center for Vascular Biology & Medicine, 975 W. Walnut Street IB 441, Indianapolis, Indiana 46202, USA. Telephone: (317) 278-0130; Fax: (317) 278-0089; e-mail: kmarch{at}iupui.edu
Received on May 21, 2007;
accepted for publication on September 10, 2007.
First published online in STEM CELLS EXPRESS September 27, 2007.
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ABSTRACT |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosure of Potential...AcknowledgmentsReferences |
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Recently it was discovered that pluripotent cells, which exhibit stem cell properties, reside in the stromal compartment of adipose tissues [12]. These adipose stromal (stem) cells (ASCs) not only possess the ability to differentiate into multiple mesenchymal cell types [12–15] but also secrete significant levels of many potent growth factors and cytokines, including vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) [8, 16]. Based on these studies demonstrating the ability of ASCs to promote endothelial cell (EC) survival as well as to enhance vascular supply in vivo, we have suggested that paracrine factor secretion is a primary mechanism of the effects of ASCs, much as was described for endothelial progenitors [17, 18].
We are presently systematically elucidating the contribution of specific secreted factors to the potency of ASCs. Initially we have focused on HGF because of its many potentially beneficial functions and recent findings that HGF may be the dominant angiogenic and protective factor secreted by ASCs [16]. HGF is a growth and motogenic factor for diverse cell types, including endothelial and smooth muscle cells, which also possesses potent angiogenic effects [19, 20]. Previously, HGF has been studied for "therapeutic angiogenesis" both via direct gene transfer [21–25] as well as approaches using cells transduced with vectors expressing these genes [26, 27]. It recently has been reported that treatment with bone marrow-derived mesenchymal stem cells overexpressing HGF could improve functional parameters of ischemic myocardium [26]. The secretion of HGF by both ASCs and adipose tissue has been described as particularly important in vasculature [28]. Thus, the multiplicity of cellular targets suggests that HGF plays a particularly significant role in the paracrine function of ASCs.
To specifically evaluate the importance of HGF for ASC-mediated contributions to revascularization both in vitro and in vivo, we employed RNA interference knockdown of HGF using lentiviral delivery of a small hairpin RNA (shRNA), based on a small interfering RNA sequence [29] that had been shown previously to selectively target HGF and downregulate its expression [30]. Our studies have demonstrated a requirement for HGF secretion in mediating the effects of human ASCs on microvascular EC growth and viability as well as promoting their angiogenic sprout formation. Extension of this approach, employing an integrating lentiviral-shRNA HGF vector for in vivo testing in the hindlimb ischemia model in nude mice, revealed a marked reduction in the angiogenic effects of ASCs with silenced HGF secretion when compared with ASCs transduced with control constructs. This study demonstrates that the proangiogenic, prosurvival, and repair-promotion activities of ASC in tissue ischemia are significantly mediated by HGF.
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MATERIALS AND METHODS |
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Construction of Lentiviral RNA Interference Vectors
The double-stranded hairpin loop-containing sequence of the shRNA used in this study was 5'-GTATCCTCACGAGCATGACAAGGCC TCAGTCATGCTCGTGAGGATAC-3', where the nonhairpin region corresponds to human HGF mRNA [30] (as italicized). The shRNA was cloned into the expression vector pND-776 (E. Rosen, unpublished manuscript) using the ApaI 5'/EcoRI 3' cloning sites in the polycloning region. This vector also contains sequences encoding green fluorescence protein (GFP) downstream of the multiple cloning sites. Expression of the shRNA was driven by promoter U6, whereas GFP expression derived from a separate cytomegalovirus promoter. The parent vector, which expresses a nonspecific RNA derived from the multicloning sequence, served as the negative control.
The expression cassettes were cloned between the Asp198 and XhoI sites of pENTR-1 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). The resulting plasmid was cloned into pcDNA-HIV-CS-CGW (a gift from Phil Zoltick, Children's Hospital, Philadelphia).
Lentiviral Vector Transductions
Lentiviral stocks were produced by transient transfection of HEK293T cells (American Type Culture Collection, Manassas, VA, http://www.atcc.org) as described previously [31–37]. A total of 5 x 106 cells were seeded in 75-cm2 flasks 24 hours before transfection. Transfection was performed by calcium phosphate precipitation method (Profection kit; Promega, Madison, WI, http://www.promega.com) according to the manufacturer's instructions. Plasmids used were the pcDNA-HIV-CS-CGW derived vectors (18 µg), pMDLg (6.6 µg), pRSV/REV (3.3 µg; Cell Genesys, Foster City, CA, http://www.cellgenesys.com), and pMD.G (4.6 µg). The vector-containing supernatants were harvested 48 hours after transfection, filtered through a 0.45-µm syringe filter, and then stored at –80°C. To transduce ASCs with lentivirus constructs, passage 1 cells were seeded at a density of 104 cells per cm2 in 6-well plates. Various volumes (50–400 µl) of lentivirus suspension and 8 µg/ml polybrene were added to EBM-2 (Cambrex) with 5% FBS in a total volume of 1 ml. Cells were allowed to incubate at 37°C for 12 hours before removing the medium and replacing it with 2 ml of fresh EGM-2MV for expansion of transductants.
Flow Cytometric Characterization of Human ASCs
Transduced GFP-expressing ASCs were detached with 0.05% Trypsin-EDTA and washed with phosphate-buffered saline (PBS), and then 2x104 cells were fixed in 2% paraformaldehyde (Tousimis, Rockville, MD, http://tousimis.com) and analyzed by flow cytometry on a FACSCalibur instrument (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) to evaluate transduction efficiency based on GFP-positive signal. Populations of transduced ASCs resulting from the use of the least volume of lentivirus stock yielding >99% GFP+ cells were used in the studies described below.
Expression of the surface marker CD31 was also analyzed in some experiments. Cells were incubated for 20 minutes with CD31-PE (BD Biosciences, San Diego, http://www.bdbiosciences.com) or matching isotype controls at a final concentration of 5 mg/ml. The antibody-labeled cells were subsequently washed with 2% FBS in PBS and fixed with 2% paraformaldehyde for analysis using a Calibur flow cytometer analyzer and Cell Quest Pro software (Becton Dickinson).
Enzyme-Linked Immunosorbent Assay Analysis of HGF Level in Human ASC Supernatants
After the fourth passage, ASCs transduced with either of the lentivirus vectors or untransduced ASCs were seeded at a density of 104 cells per cm2 in a 6-well culture dish and grown in EBM-2 with 5% FBS (EBM-2/5% FBS) at 37°C in 5% CO2. After overnight attachment, the medium was changed and the cells were incubated an additional 24 hours before the media were collected and cells removed by centrifugation at 300g for 5 minutes for enzyme-linked immunosorbent assay (ELISA) analysis. Each well of a 96-well ELISA plate (catalog number 3585; Corning Costar, Acton, MA, http://www.corning.com/lifesciences) was coated with 0.1 µg of a mouse monoclonal HGF capture antibody (MAB 694; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and incubated for 1 hour at room temperature. Nonspecific binding sites were blocked with 300 µl of PBS containing 1% bovine serum albumin, 5% sucrose, and 0.05% NaN3. Supernatants or purified HGF (294HG; R&D Systems) standard (100 µl) at the appropriate dilutions were added to wells in triplicate and incubated for 2 hours before adding 100 µl of the biotinylated detection antibody (anti-mouse IgG; R&D Systems; BAF 294, 200 ng/ml) for 2 hours. After washing, 100 microliters per well streptavidin conjugated horseradish peroxidase (HRP; DY998; R&D Systems), diluted 1:200, was added, and the plate was incubated for 20 minutes, followed by three washes, after which 100 microliters per well Substrate Solution (H202 and DY999; R&D Systems) was added, and the plate was incubated for 20–30 minutes before adding 50 microliters per well Stop Solution (1 M H2SO4). The optical density of each well was assayed at 450 nm and corrected for the background light absorption. VEGF in cell supernatants was determined using commercial ELISA kits (human VEGF Quantikine; R&D Systems).
The Effect of Serum Deprivation on the Survival of Transduced ASCs
Human adipose stromal cell-small hairpin control (ASC-shCtrl) and adipose stromal cell-small hairpin hepatocyte growth factor (ASC-shHGF) were seeded in EGM-2MV at 5,000 cells per well in 96-well plates and incubated overnight. The medium was replaced with EBM-2 with or without 5% FBS, and the cells were incubated for 12 hours. Cell viability was determined using the CellTiter 96 AQueous One Solution Reagent (number G358; Promega) according to the manufacturer's protocol. Data are expressed as relative fluorescence per 1,000 cells.
Human Microvascular Endothelial Cell Proliferation Assays
Human microvascular endothelial cells (HMVECs) were purchased from Cambrex (cc-7030) and cultured in EGM-2MV medium. Cells were used within three passages of initial thawing. To assay the effects of conditioned media on growth, HMVECs were seeded at a density of 5,000 cells per cm2 in 12-well plates (Techno Plastic Products AG, Trasadingen, Switzerland, http://www.tpp.ch). HMVECs were incubated for 24 hours with 2 ml of fresh EBM-2/5% FBS medium, which supports viability but not growth or migration of these cells. For cell migration and proliferation studies, an equal volume of EBM-2/5% FBS medium harvested from ASCs was added to the medium and the cells were cultured. For some experiments, the ASC-conditioned medium was preincubated for 1 hour with 1 µg/ml anti-human HGF neutralizing antibody (AF-294-NA; R&D Systems). Cells were detached after 72 hours and cell numbers counted using 0.4% trypan blue exclusion to monitor viability. Individual experiments were performed in triplicate wells and repeated at least three times.
Quantitation of In Vitro Sprout Formation by HMVECs
Angiogenesis in vitro was tested as described previously [34]. Briefly, the HMVECs were grown to confluence on cytodex-3 microcarrier beads (C-3275; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) whose diameter is 133–215 µm, and approximately 100 beads were placed into each well of a 12-well culture plate coated with 0.5 ml of 2.5 mg/ml fibrinogen (F-4883; Sigma) in PBS that had been polymerized by adding 0.48 U/ml thrombin (T-7009; Sigma). Approximately 1 ml of either the conditioned medium, EGM-2MV (positive control), or EBM-2/5% FBS (negative control) was added to each well, and the chambers were then incubated at 37°C, 5% CO2. After 1 hour, the medium was exchanged and the plates were incubated for an additional 48 hours. For quantitation, only the sprouts that were longer than the diameter of the average bead size were counted using a phase contrast microscope. Experiments were performed in triplicate and repeated at least three times.
Transwell Chamber Migration Assays
A Falcon cell culture insert system (Becton Dickinson) along with a compatible 24-well Falcon tissue culture plate were used for migration assays. The polyethylene terephthalate membrane pore size of 8 µm was selected to allow passage of high proliferative potential human umbilical vein-derived endothelial progenitor cells (EPCs) [35]. The membrane was precoated with 60 µl of a 50 µg/ml solution of type I collagen. The bottom chamber contained 600 µl of conditioned medium, in some cases with purified HGF or the HGF neutralizing antibody. The top chamber was seeded with 3 x 104 EPCs at the beginning of the experiment. Migration assays were terminated after 4 hours, and EPCs that had migrated through the membrane were then stained (after removal of cells remaining on top with a wet Q-tip) using Diff-Quik staining kit (Dade Behring, Newark, DE, http://www.dadebehring.com) according to manufacturer's instructions. Stained cells in 3–5 micrograph fields, obtained at x400 magnification, were counted manually. Results presented are from three independent experiments with duplicate wells for each condition.
Mouse Hindlimb Ischemia Model
The animal studies were approved by the Indiana University School of Medicine Animal Use and Care Committee. Unilateral hindlimb ischemia was created in 8-week-old male nude mice as described previously [36, 37]. The animals were anesthetized by isoflurane inhalation. An incision was made at the midline of the left hindlimb. The femoral artery and its branches were ligated, beginning from the inguinal ligament to the bifurcation of saphenous and popliteal arteries. The region between the ligatures was excised. The incision was closed with 6–0 silk sutures (Ethicon, Somerville, NJ, http://www.ethicon.com).
After surgery, the mice were randomly separated into three groups (n = 10 per group). One day after creation of unilateral hindlimb ischemia, 106 ASC-shHGF or ASC-shCtrl in 100 µl saline or saline only were injected through the tail vein using a 0.3-cc insulin syringe and a 28-gauge needle.
Blood flow restoration in ischemic limbs after treatment was evaluated by laser Doppler perfusion imaging (Moor Instruments, Devon, U.K., http://www.moor.co.uk) as described previously [8]. Briefly, animals were anesthetized by isoflurane inhalation and placed on a heating pad set at 37°C. Data were collected from the plantar surfaces of both limbs. Measurements were performed on days 1, 5, 10, 15, and 20. The results are expressed as the ratio of perfusion in ischemic (left) to nonischemic (right) limbs.
Histological Analysis
Three mice from each group were sacrificed at 1 day and the rest at 20 days after cell infusion. Gastrocnemius muscles of both limbs were removed, fixed with 10% neutrally buffered zinc formalin, and embedded in paraffin. Thin sections (5 µm) were probed with biotinylated antibodies to CD31 (550274; BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) and GFP (632380; Clontech, Palo Alto, CA, http://www.clontech.com). Visualization of binding was accomplished by streptavidin-HRP complex formation and followed by color development with 3,3'-diaminobenzidine (Sigma). Images of sections were obtained on a Nikon (Tokyo, http://www.nikon.com) microscope (model TE2000-S) and were analyzed with Image J software (National Institutes of Health, Bethesda, MD, http://www.nih.gov). Five fields from each muscle section were randomly selected for quantification of CD31 (x400 magnification).
Fluorescence Imaging of Tissue Sections
Slide-mounted, fixed, and paraffin-embedded muscle thin sections (5 µm) were preincubated with M.O.M. (BMK-2202; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) mouse Ig blocking reagent before incubating overnight with the mouse monoclonal GFP antibody (diluted 1:300) and rabbit anti-smooth muscle 
-actin (RB-9010-P0; diluted 1:50 Lab Vision Corp., Fremont, CA, http://www.labvision.com). Slides were washed and then incubated with fluorescein-conjugated chicken anti-mouse IgG (A21200
[GenBank]
; Invitrogen) and goat anti-rabbit IgG (T2769; Invitrogen) for 30 minutes. Finally, sections were incubated with 4',6-diamidino-2-phenylindole dihydrochloride (D8417; Sigma) for 1 minute. Images of sections were obtained at x400 magnification and were analyzed with Image J software.
An extensive quantitation of GFP+ ASCs present in muscle tissues at 1 and 20 days was also performed. The entire muscle was sectioned into 10–15 thin sections, each separated by 100-µm intervals, and then 10 randomly selected fields (x200 magnification) from each section were quantitated.
Statistical Analysis
Data are expressed as mean ± standard deviation or standard error of the mean, as noted in the text and figures. Statistical comparisons between groups were performed with a two-tailed Student's t test. Comparisons of multiple groups were done with analysis of variance with corrections for multiple comparisons; p < .05 was considered significant.
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RESULTS |
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–4). Although ASCs cultured in EBM-2 produce all of the supplemented protein factors present in EGM-2MV, most of these accumulate in the medium to substantially lower levels than what is added to EGM-2MV [38]. In addition, ASCs may also secrete factors that negatively influence the performance of endothelial cells in the assays employed in this study.
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Silencing HGF Compromises the Ability of ASC to Support Angiogenesis In Vitro
The consequence of reduced HGF expression was further functionally analyzed by determining the ability of the various conditioned media to stimulate angiogenic sprout formation by HMVECs in vitro (Fig. 3; representative image of HMVEC forming sprouts is shown in 3A). Sprout formation was 2.1-fold higher when cultured in the CM from untransduced ASCs (1.82 ± 0.12 sprouts per bead) and ASC-shCtrl transduced with control shRNA (1.82 ± 0.26 sprouts per bead) compared with EBM-2/5% FBS (0.88 ± 0.13 sprouts per bead, p < .01). Stimulation was nearly absent when an equivalent amount of ASC-shHGF CM was used (1.04 ± 0.09). Thus, angiogenic stimulation by ASCs is also greatly compromised by silencing HGF expression.
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The Persistence of ASCs in Ischemic Tissues Is Influenced by HGF Levels
The influence of HGF expression on the ability of systemically delivered ASCs to home to ischemic tissues and persist during repair and reperfusion of tissues was determined by quantitating GFP+ cells in the lower legs at 1 and 20 days after delivery. Green fluorescing ASCs could be detected in both normal and ischemic tissues of the lower hindlimb (Fig. 7A); however, at 1 day after infusion, both ASC-shHGF and ASC-shCtrl accumulated to a fivefold greater density in ischemic tissues compared with normal tissues. These data indicate that the secretion of HGF did not influence homing of ASCs to sites of ischemic injury.
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Stably Engrafted ASCs Are Often Located in Proximity to Regenerating Myofibers
The spatial location of GFP-expressing ASCs within thin sections of ischemic tissues harvested at 20 days after cell infusion was further explored by fluorescence microscopy. Accurate quantitation of fluorescence in situ can be difficult due to autofluorescence of connective tissues; therefore, we also probed the tissues with smooth muscle -actin, which is expressed by ASCs [12], in addition to arterioles (Fig. 7B). It was often observed that the ASCs had engrafted adjacent to skeletal myofibers containing centrally located, uncondensed nuclei, which is a hallmark of regenerating myofibers [38]. Groupings of ASCs were also visible around mature, -actin-positive vessels (presumably arterioles).
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DISCUSSION |
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Identifying functional mechanisms of cell therapy is important in that it defines the boundaries which govern potential therapeutic applications. The complementary, but not mutually exclusive, concepts of therapeutic cells having "manager" or "software" functions versus providing "building blocks" or "hardware" [39, 40] are important in that they define the technical limits for therapeutic applications of cells. The near-term prospects for translating cell therapies to the clinic would appear to be much greater if the therapeutic effect is primarily through paracrine signaling to enhance endogenous repair mechanisms. This concept directly builds on earlier attempts to develop gene and protein therapeutic modalities but has the potential for enhanced benefit over monotherapies due to the ability of viable cells to deliver a milieu of beneficial growth factors and cytokines to the site of injury. Specific targeting may occur through chemoattraction to the injury site as a result of injury-enhanced cytokine release combined with cell receptor-mediated anchoring to exposed ligands. Selective engraftment could confer additional advantages over other delivery vehicles by potentially reducing stimulation of unwanted cell growth resulting from nonselective exposure to nontarget tissues, which would be particularly important for broad-acting molecules such as HGF.
Although the potential medical benefits of regenerative cellular therapy are enormous, it is evident that achieving productive and directed differentiation of pluripotent cells for repair of tissues will certainly require much more research into the exact mechanisms controlling cellular plasticity in order to adequately address issues of both safety and efficacy. On the other hand, delivery of "mini-drug factories" in the form of cells is comparatively free of technical hurdles and is associated with fewer safety concerns for treating a wide variety of acute and chronic disorders. This would be especially true for cells such as ASCs that can be isolated in high abundance and used autologously with minimal manipulation.
ASCs secrete a plethora of cytokines and growth factors [8], yet the ability of the cells to promote angiogenesis when exposed to acute ischemia is substantially abolished by suppressing levels of a single gene product (HGF). Many of the other factors secreted by ASCs also have well-defined angiogenic effects, and certainly silencing factors, such as VEGF, would result in reduced angiogenic potential of ASCs. It is also possible that enhancing levels of VEGF or other potent growth factors could even overcome the defect cause by HGF silencing. We are presently testing many of these possibilities; presently, however, we chose to specifically silence HGF because it is one of the most abundant proteins secreted by ASCs, and there is a substantial body of literature demonstrating clear and consistent broad spectrum benefits of exogenously added HGF in many models [41–43]. It is also clearly evident that HGF has critical functions in the endogenous pathophysiological processes inherent to diseased or injured tissues [44, 45].
Secretion of HGF in response to injury or disease appears to promote survival of cells in affected tissues as well as promote repair through attracting stem and progenitor cells. There is substantial evidence that HGF is a key molecule signaling the mobilization, migration, and homing of endogenous stem and progenitor cells originating in various tissue depots [46, 47]. Pathologically induced upregulation of HGF may recruit endogenous progenitor and stem cells to replace damaged tissues. Pluripotent cells from the bone marrow and blood constitutively express functional HGF receptor (c-Met) and are induced to migrate and differentiate in response to HGF gradients [46–49]. Populations of these cells possess the ability to differentiate into vascular and mesenchymal cell lineages [46, 50–52].
Numerous groups have demonstrated the plasticity of ASCs in vitro; thus, it is possible that, in addition to providing paracrine support, ASCs also contribute directly to repair through transdifferentiation and incorporation into regenerating skeletal muscle tissues. Although we cannot conclusively rule out differentiation of ASCs into myocytes and vascular cells, it appears that the predominant effect of ASCs, at least when provided systemically, is to support endogenous repair or limitation of ischemic damage. Immunofluorescence micrographs of thin sections revealed that ASCs within ischemic tissues were often localized in juxtaposition to arterioles and myofibers, of which the latter were undergoing active regeneration, as indicated by centrally located, uncondensed nuclei (Fig. 7B). To determine the temporal relationship between ASC engraftment and tissue regeneration, it will be necessary to conduct a more extensive time course to determine whether ASCs specifically home to the region of damaged myocytes and accelerate repair or whether chance localization of these cells promotes repair. These studies, as well as studies addressing transdifferentiation (or even fusion) events, are currently in progress.
Another intriguing hypothesis is that an autocrine loop exists in c-Met-expressing ASCs [53]. Given the antiapoptotic effects of HGF, it is possible that silencing its expression disrupts this intracellular survival mechanism, making the cells expressing shRNA targeting HGF less fit for survival in hostile ischemic environments, as suggested by the in vitro experiments with serum deprivation (Fig. 1G). In fact, although equivalent numbers of both cell types are present in ischemic muscles immediately after infusion, reduced HGF expression by ASC-shHGF causes these cells to be cleared more rapidly during prolonged ischemia compared with ASC-shCtrl cells (Fig. 7). We are presently conducting experiments to determine whether a functional autocrine loop exists in these cells and whether reduced or enhanced signaling through HGF influences the survival of ASCs administered in vivo.
In conclusion, we and others have demonstrated the therapeutic potential of ASCs for promoting revascularization of ischemic tissues. These cells appear to have unique properties that make them attractive candidates for autologous cell therapy. Precisely because of these properties, it is possible to anticipate positive outcomes in imminent clinical trials in patients with peripheral arterial disease.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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, p < .05 vs. ASC-shHGF). Abbreviations: Ab, antibody; ASC, adipose stromal cell; EBM-2, endothelial basal medium-2; EGM-2MV, endothelial growth medium-2 MV; HGF, hepatocyte growth factor; shCtrl, small hairpin RNA targeted to an inactive control sequence; shHGF, small hairpin RNA targeted to hepatocyte growth factor mRNA.
