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. Author manuscript; available in PMC: 2013 Feb 15.
Published in final edited form as: Exp Cell Res. 2011 Dec 13;318(4):416–423. doi: 10.1016/j.yexcr.2011.12.002

Long-term In-Vivo Tumorigenic Assessment of Human Culture-expanded Adipose Stromal/Stem Cells

Zoe Marie MacIsaac a, Hulan Shang b, Hitesh Agrawal b, Ning Yang b, Anna Parker c, Adam J Katz b
PMCID: PMC3264753  NIHMSID: NIHMS344919  PMID: 22185824

Abstract

After more than a decade of extensive experimentation, the promise of stem cells to revolutionize the field of medicine has negotiated their entry into clinical trial. Adipose tissue specifically holds potential as an attainable and abundant source of stem cells. Currently undergoing investigation are adipose stem cell (ASC) therapies for diabetes and critical limb ischemia, among others. In the enthusiastic pursuit of regenerative therapies, however, questions remain regarding ASC persistence and migration, and, importantly, their safety and potential for neoplasia. To date, assays of in vivo ASC activity have been limited by early end points. We hypothesized that with time, ASCs injected subcutaneously undergo removal by normal tissue turnover and homeostasis, and by the host’s immune system. In this study, a high dose of culture expanded ASCs were formulated and implanted as multicellular aggregates into immunocompromised mice, which were maintained for over one year. Animals were monitored for toxicity, and surviving cells quantified at study endpoint. No difference in growth/weight or lifespan was found between cell-treated and vehicle treated animals, and no malignancies were detected in treated animals. Moreover, real-time PCR for a human specific sequence, ERV-3, detected no persistent ASCs. With the advent of clinical application, clarification of currently enigmatic stem cell properties has become imperative. Our study represents the longest duration determination of stem cell activity in vivo, and contributes strong evidence in support of the safety of adipose derived stem cell applications.

Keywords: Adipose derived stem cell, Mesenchymal Stem Cell Transplantation/toxicity

Introduction

Due to their potential impact on the field of medicine, adipose stem cells (ASCs) have received extensive attention and publicity since their description in 2001 [1]. Retaining the ability for multipotentiality and capable of prodigious environmental influences, ASCs, unlike their counterparts, are both accessible and abundant [1–5]. With anticipation, ASCs have been introduced into clinical trial, despite an incomplete understanding of their mechanisms. The cells’ in vivo migration patterns and persistence remain unclear [6]. Attempts thus far to uncover these unknowns have been hindered by short lived assays, and, frequently, utilization of non-human cells, which often exhibit traits differing from human derived cells. Even more concerning than the cells’ indeterminate fate is the question of their safety [7]. The only report of adipose stem cell associated spontaneous malignancy has been retracted [8], but other stem cell types/sources, most notably embryonic, become tumorigenic under certain conditions [9, 10]. Numerous centers have expanded and applied the cells without report of toxicity for over a decade, but a more definitive answer to the question of malignancy is critical as adipose stem cells enter clinical testing/utilization, on their way to becoming established therapeutic options [11, 12].

In this experiment, human adipose derived stem cells were culture expanded and injected subcutaneously as multicellular aggregates at high doses into immunocompromised mice. Treated animals were maintained for more than one year alongside vehicle-treated controls. Animals were systematically examined for illness, and growth was compared by serial weight. At time of harvest, final weights were recorded and organs examined for evidence of tumor formation. Lungs and spleens weights were recorded, and livers were sectioned and examined for gross and microscopic lesions. Real-time PCR for ERV-3, a primate-specific 130 bp retrovirus present in known quantity in human cells, was utilized to detect and quantify persistent ASCs in select organs identified as primary sites of metastasis after subcutaneous tumorigenic application [13–16], as well as the injection site itself.

Materials and Methods

Isolation and culture expansion of human adipose stem cells (ASCs)

Human adipose tissue samples were obtained from elective surgical procedures under IRB approval at the University of Virginia Health System and immediately transported to the laboratory. ASCs were isolated as previously described [17, 18]. Briefly, samples were washed, enzymatically dissociated, and filtered to remove debris [18]. After centrifugation, pelleted cells were recovered and washed. Contaminating erythrocytes were removed by osmotic buffer, and the cells were plated onto tissue culture plastic and culture-expanded in adherent monolayer culture in xenogeneic-free growth medium with 1% human serum (LM1%). After three passages, culture-expanded ASCs were placed on the inside of a culture dish lid in forty-microliter droplets, and the lid was inverted to induce MA formation using the hanging droplet method [19–21]. Cells in the resulting hanging droplets were allowed to form 3D, self-assembling spheroids, reaching 40,000 cells per aggregate over the course of two weeks (“Group 1”). A second group of culture-expanded ASCs were cryopreserved before spheroid formation (“Group 2”). After thawing, these cells were maintained in suspension culture for 5 weeks prior to implantation, also reaching 40,000 cells per aggregate.

ASC implantation into an in vivo, immunocompromised model

After obtaining approval from the IACUC at the University of Virginia, twelve athymic Ncr-nu/nu mice (National Cancer Institute) were anaesthetized using isoflurane and injected subcutaneously with 6 million ASCs each (150 ASC spheroids, each composed 40,000 ASCs), followed by appropriate postoperative pain control. Six control animals were administered empty vehicle injections. Over 12 months, weights were serially recorded and animals monitored for evidence of illness and/or gross tumor growth. Upon manifestation of illness, or at experimental end point, animals were weighed and sacrificed. Organs were harvested and evaluated for gross appearance, and lung and spleen weights recorded.

Quantification of human adipose stem cells

Real-time PCR for ERV-3 was used to evaluate the migration and persistence of human ASCs. The primers for the human-specific ERV-3 gene were designed as previously described [22, 23]: forward, 5-ATG GGA AGC AAG GGA ACT AAT G; reverse, 5-CCC AGC GAG CAA TAC AGA ATT T (Integrated DNA Technologies). Samples of the entire spleen, lung, and from the injection site were frozen with liquid nitrogen and ground to a powder using mortar and pestle. DNA extraction was performed with DNAzol (Molecular Research Center) according to manufacturer’s protocol. DNA extract from cultured ASCs served as a positive control (i.e. ASCs 100%), and DNA extract from an untreated mouse was used as a negative control (i.e. ASCs 0%).

Standards were prepared by combining cultured ASCs and mouse tissue in variable ratios of cell numbers (ASCs: 4.76%, 0.498%, 0.05%, and 0.005%). Accordingly, standard ASCs originated from (5×104, 5×103, 5×102 and 5×101) mixed with 106 mouse embryonic fibroblast cells. Genomic DNA was extracted from these preparations according to experimental protocol. Real-time quantitative PCR with 96-well optical plates was performed and analyzed using an iCycler iQ (BioRad). Each reaction was performed using 1.5 ul of diluted DNA, 6.25 ul of SYBR green (BioRad), 0.5 ul forward and reverse primers, and 3.75 ul of PCR-grade H2O. Extracted DNA was assessed for quality and quantity using a GeneQuant Pro (Amersham Biosciences) and each sample run at 1:10 and 1:50 dilutions in duplicate. The PCR conditions used were: step 1: 95°C for 15min.; step 2: 45 cycles, each with 30s at 95°C (denaturation), 30s at 60°C (annealing) and 30s at 72°C (extension). Threshold cycle (CT) was defined as the first cycle number in a PCR amplification above baseline and during the exponential increase period, with 40 as the maximum allowable value. Appropriate amplification was determined by melt curve analysis, ERV-3 melting temperature 87.5°C.

Histological evaluation

Histological evaluation was selectively performed on animals that died prior to the study endpoint or that were sacrificed early because of obvious morbidity or gross abnormality. Specifically, livers from mice with early deaths and/or abnormal findings were cryosectioned and stained with hematoxylin and eosin, with one healthy control for comparison. Slides were examined by an expert pathologist blinded to treatment.

Statistical analysis

Animal weights, organ weights and lifespan were compared using student’s t-test, p< 0.05.

Results

Animal survival

Between the two arms of this study (cultured cells, cryopreserved cells), each of 12 mice were injected subcutaneously with 6 million culture expanded adipose stem cells formulated as 3-dimensional spheroids, corresponding to a dose of 240 million cells/kg. A control group was injected with vehicle only. Of treated animals, 11 of 12 survived to experimental endpoint (one death 8 months post-injection), compared to only 3 of 6 untreated controls (deaths at 3 and 10 months post-injection, and sacrifice at 11 months due to abdominal distention). There was no significant statistical difference between treated and control group lifespan.

Animal weights

Cell treated animals received a dose of 240 million cells/kilogram. Despite the large burden of human cells, treated animals thrived, and weights in both groups followed a similar trend over time (figure 1). There was no significant statistical difference between treated and untreated animal weights at 12 months.

Figure 1. Treated and control weight over time, with standard error.

Figure 1

Figure 1

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Control and treated animal weights followed a similar trend over time in both in both (a) non-cryopreserved and (b) cryopreserved groups. There was no significant difference between treated and control group weights by student’s T-test.

Organ assessment

Upon animal death, heart, lungs, spleen, pancreas, liver, brain, and kidneys were weighed and examined for gross abnormality, in addition to examination of the injection site. Treated animals demonstrated no gross evidence of tumor formation either at the site of injection, nor in any harvested organs. In the treatment group, the only abnormality was a nodular spleen. In the untreated control group, one animal revealed markedly enlarged, nodular spleen (figure 2). This was in the animal sacrificed early due to abdominal distention. Measurement of lung and spleen weights and comparison between treated and untreated group weights revealed no significant difference for either organ.

Figure 2. Gross organ abnormalities.

Figure 2

Figure 2

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(a) Treated Ms39 with nodular spleen, (b) control Ms435 with enlarged, nodular spleen.

Histology

At time of death, animal livers were frozen and preserved. Livers from animals with early deaths and those from animals with abnormal manifestations, in addition to one normal control, were later retrieved for cryosection. Of the treated group, this included one animal with a normal sized but nodular spleen, and one animal with an early death. Of the control group, this included two animals with early deaths, one animal with hind limb paralysis, and one animal euthanized secondarily to marked abdominal distention. Three sections from three locations were taken from each organ (nine for each organ) and stained with hematoxylin and eosin. Slides were assessed by a pathologist at our institution who was blinded to treatment. Only samples from the normal control were identified as non-pathologic. All other livers displayed evidence of long-standing necrosis and/or peliosis. One malignancy, lymphoma, was identified in the control animal euthanized early due to abdominal distention (table 1, figure 3).

Table 1.

Abnormal findings

Treatment group Control group
Ms39, nodular spleen Ms44, early death
Ms42, early death Ms45, hind limb paralysis
Ms430, early death
Ms435, euthanized due to severe abdominal distention
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Figure 3. Liver histology, hematoxylin and eosin.

Figure 3

Figure 3

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(a) Control Ms435 (shown with 1 mm reference) with lymphoma (arrow), (b) Treated Ms42 (shown with 200 um reference) with evidence of peliosis (arrow), a vascular condition of unknown etiology characterized by randomly distributed blood filled sinuses throughout the liver, and necrosis.

PCR for Cell Persistence and Migration

Real-time PCR was performed using ERV-3 amplification to assess for human cell presence in lung and spleen samples, and from the site of injection. Two standards were utilized for each PCR run, one using variable concentrations of human cells mixed with mouse tissue, the other consisting of variable concentrations of human cells only. Log [ASC] linearly correlated with CT values, correlation coefficient >0.99, +/− 0.0069. Real-time PCR for ERV-3 yielded no presence of human cells in either experimental or control animal organs, nor at the site of injection (figure 4).

Figure 4. RT-PCR for ERV-3.

Figure 4

Figure 4

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Two standard curves were used, (a) one consisting of varied concentrations of human cells only, and the other (b) consisting of varied concentrations of human cells mixed with mouse tissue, either lung, spleen or skin and subcutaneous tissue. Both the human cell only standards and the relevant tissue standard curve were included in each PCR run.

Discussion

Although stem cells possess great potential for revolutionary therapies, a major concern revolves around the potential for tumorigenic activity associated with their use. This is especially pertinent to embryonic stem cells [24]. Although adult ASCs do not seem to exhibit the same tumorigenic potential, the possibility remains perhaps the most serious downside of their translation to the clinic. Oncogenesis in humans tends to evolve over extended periods of time, and statistically supported cause-and-effect relationship can require large number of subjects, making definitive claims of cell therapy safety difficult.

Nude mice have long been established as an effective model of human tumorigenesis in vivo [25–27]. The animals readily accept human tumor xenografts, and depending on the number of cells injected, develop tumors in as few as 1–8 weeks [28]. Although the animals lack certain components of the immune system, B cells, dendritic cells and granulocytes remain relatively intact, with a compensatory increase in both natural killer cells and tumoricidal macrophages [28, 29]. The ability to place of human tumor grafts orthotopically within this environment increases clinical applicability [29]. We applied our treatment subcutaneously, the likely site of application upon clinical translation. In addition to examining the injection site, we chose to analyze lung, liver and spleen based on previous studies determining the location of metastasis after subcutaneous tumor implantation [13–16].

Given the emerging interest in ASCs and their progression into clinical trials, our objective in this study was to evaluate ASC persistence, migration, and propensity for tumorigenesis over an extended time period after high dose implantation into a nude mouse model.

Previous attempts to determine the migration and persistence of stem cells in vivo include GFP labeling methods and reporter gene analysis [30–33]. These studies either noted a lack of stem cell persistence, or experienced time-dependent limits of assay efficacy [33–35].

In contrast, Vilalta et al found persistence after 8 months [36]. ASCs were transfected with GFP reporter genes and maintained in culture for 2 months, then injected into nude mice at a dose of 5×105 cells/animal. After 1 week and throughout the remaining months, 1.5% of cells applied intravenously remained, located within the liver. Of intramuscularly applied cells, 75% remained locally, with a limited number also detectable in the liver. No toxic side effects were observed, and at 8 months, GFP-labeled ASCs were retrieved from muscle and cultured. The investigators were unable to retrieve ASCs from the liver [36].

RT-PCR for ERV-3 holds advantages over GFP-bioluminescence and other methods of in vivo stem cell detection. Both sensitive and specific, RT-PCR for ERV-3 detects as few as 5 human cells in 1,000,000 murine cells (0.005%) [22, 23], compared to detection of 5,000 cells/organ reported in GFP-studies [31]. RT-PCR for ERV-3 detects human cells without need for cellular manipulation. The assay remains effective across time, and will detect even human cell remnants, as detection is not dependent on cellular viability.

The application of multicellular aggregates containing 6 million cells represents an especially aggressive formulation. Compared to the highest dose currently in clinical trial, 5.7 million ASCs/kg [37], we applied 240 million ASCs/kg, a 42 fold higher dose. Moreover, application as multicellular aggregates (MAs) would potentiate tendencies toward malignancy. MAs accurately reproduce in vivo tumor microenvironments [21, 38]. Cultured in hanging drops, cells form highly organized, three-dimensional, tissue like structures with extensive extracellular matrix, retaining longer viability and functionality [19–21], and are robust enough for easy maintenance in serum free culture [19]. While only 5×102 human colon carcinoma cells in multicellular aggregate establish malignancy in nude mice, as many as 2×106 non-aggregate carcinoma cells remain ineffective [38], and multicellular aggregates are heavily utilized as models for tumorigenesis [21, 38]. That our aggressive formulation produced no tumor over the course of the nude mouse lifespan speaks highly to the safety of these cells.

Our results differ from those of Vilalta et al, and are most applicable to cells expanded for up to 3 weeks and delivered by subcutaneous injection. The difference between those results and ours may be the duration of expansion (several weeks vs. 2 months), cellular formulation (multicellular aggregate vs. single cell expansion), method of injection (subcutaneous vs. intramuscular and intravenous injection) and ASC manipulation (no manipulation vs. genetic transfection).

Our study represents the longest-duration determination of stem cell persistence, one year, at which point no ASCs were detected. Both components of the nude mouse immune system, ASCs are susceptible to lysis by T- and NK cells [6]. Consistent with a previous study, where TUNEL assay ruled out dissolution by apoptosis, the cells may well survive long enough to exert therapeutic effect and gradually undergo removal by the host immune system, as well as by normal tissue turnover and homeostasis [35].

Future experiments could examine other tissues presence of ERV-3. Additionally, organs could be harvested at earlier dates for ERV-3 detection to determine ASC patterns of migration. A useful adjunct to this would be immunolabeling for imaging of ASC migration patterns. It will be important to establish the long term persistence of ASCs following other methods of culture expansion in order to expand the clinical applicability of these results.

Conclusion

As stem cells approach translation to the clinic, animal models remain a critical first-pass tool with which to scrutinize stem cell safety profiles. Cells cannot be labeled when destined for human-application, and human genomic DNA and transcription cannot differentiate the applied cells within human hosts. The decade of experience of laboratories culturing and applying adipose stem cells without toxic report provides powerful evidence that ASCs may be safely applied for medical therapies. As a specific test for toxicity and tumorigenic potential, this study applied a heavy burden of ASCs in the form of potent multicellular aggregates. The results contribute further evidence that human culture-expanded ASCs do not generate tumors after subcutaneous implantation in vivo at high doses, when formulated as 3-D organoids composed of cells and self-generated ECM.

Table 2.

Real Time-PCR results for mouse injection site, lung, spleen

Group1 Injection site Lung Spleen Group2 Injection site Lung Spleen
Control 1 ND ND ND Control 1 ND ND ND
Control 2 ND ND ND Control 2a ND ND ND
Control 3a ND ND ND Control 3a ND ND ND
Implanted 1 ND ND ND Implanted 1 ND ND ND
Implanted 2 ND ND ND Implanted 2 ND ND ND
Implanted 3 ND ND ND Implanted 3 ND ND ND
Implanted 4 ND ND ND Implanted 4 ND ND ND
Implanted 5a ND ND ND Implanted 5 ND ND ND
Implanted 6 ND ND ND Implanted 6a ND ND ND
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ND: no detection for human cells in the tissue

a

Died earlier than a year

Table 3.

Previous studies for ASC persistence and migration

Passages cultured Method of detection Method of application Findings
Altman et al, 2009 [39] 1 to 8 GFP (lentiviral vector) Seeded on a silk fibroin-chitosan scaffold which was sutured to full thickness defects in athymic male mice Week 4: ASCs survived and differentiated into epidermal epithelial cells, fibrovascular cells, and endothelial cells. No migration was detected.
Bai et al, 2010 [40] 3 GFP/Luciferase fusion gene (lentiviral vector) 5×105 cells injected into the peri-infarct region of 8–12 week old immunocomprimized mice Week 8: ASCs survived in the infracted myocardium. 3.5% differentiated into cardiomyocytes or endothelial cells. No migration was detected.
Lalande et al, 2011 [41] Labeling with iron oxide, detected by magnetic resonance imaging 5×104–5×105 cells seeded on a porous polysaccharide-based scaffold Week 4: ASCs remained detectable and migrated to the area surrounding the implant.
Levi et al, 2010 [36] 1 Fluorescent in situ hybridization for human sex chromosomes PCR for hGAPDH, hALP, hRUNX2 1.5×105 cells seeded on poly(lactic-co- glycolic acid) scaffolds applied to full thickness parietal defects in nude mice Week 4: No ASCs were detectable by fluorescent in situ hybridization.
Week 8: No ASCs were detected by PCR.
Meyerrose et al, 2007 [42] 4 GFP (retroviral vector) PCR for GFP transgene and human Alu repeats 106 cells applied intravenously, intraperitoneally, or subcutaneously to sublethally irradiated and nonirradiated immunocomprimized mice Days 35, 45, 60 and 70: (+)ASCs were detected in the intestine, heart, spleen, liver, lung, kidney, muscle, brain or fat, with a decreasing trend over time. There was no extensive proliferation at site of lodgment.
Vilalta et al, 2008 [43] 12 GFP/Luciferase fusion gene (lentiviral vector) 5×105 cells applied intramuscularly or intravenously to nude mice 8 months: 75% of intramuscularly applied cells remained at the site of injection, with a limited number in the live.r Of intravenously applied cells, a “limited number” were detected within the liver
Wolbank et al, 2007 [34] 6a Red luciferase 105 cells/gm animal weight applied intraperitoneally, intramuscularly or subcutaneously, or 106 cells within a fibrin matrix implant applied to nude mice Week 3: Some ASCs were “located ventrally” when applied without fibrin. There was no migration when applied with high fibrin concentration
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a

followed by either 3 weeks of osteogenic or 3 passages of adipogenic differentiation

Highlights.

  • Adipose stem cells promise novel clinical therapies.

  • Before clinical translation, safety profiles must be further elucidated.

  • Subcutaneously injected non-autologous adipose stem cells do not form tumors.

  • Subcutaneously injected non-autologous adipose stem cells undergo complete removal by one year.

Acknowledgments

Funding: Funded in part by the U.S. Army Medical Research and Materiel Command (USAMRMC) as part of the Armed Forces Institute of Regenerative Medicine (AFIRM) (AK), and by NIH/NIBIB Grant R21 EB009140 (AK)

We would like to thank Christopher A. Moskaluk M.D., PhD, University of Virginia Department of Pathology, for his assistance with histological analysis, and also Laurie Meszaros, PhD, University of Pittsburgh, for her assistance

Footnotes

Disclosure of potential conflicts of interest: Dr. Adam Katz receives royalties from patents related to adipose stem cells.

Adipose Stem Cell (ASC), Xenogeneic-free growth medium with 1% human serum (LM1%), Autologous bone-marrow derived stem cells (BMSCs), Fluorescence in situ hybridization (FISH), Real time PCR (RT-PCR), Multicellular aggregates (MAs), Threshold cycle (CT), Green fluorescent protein (GFP)

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Contributor Information

Zoe Marie MacIsaac, Email: zmm4a@virgina.edu.

Hulan Shang, Email: shanghulan@gmail.com.

Hitesh Agrawal, Email: hiteshdos@hotmail.com.

Ning Yang, Email: ny6u@virgina.edu.

Anna Parker, Email: amp4v@virginia.edu.

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