Human adipose-derived stromal/stem cells demonstrate short-lived persistence after implantation in both an immunocompetent and an immunocompromised murine model
© Agrawal et al.; licensee BioMed Central. 2014
Received: 20 April 2014
Accepted: 8 December 2014
Published: 18 December 2014
Mesenchymal cells are emerging as a promising cell platform for regenerative therapies. However, the fate of cells after transplantation in many different disease settings and tissue beds remains unclear.
In this study, human adipose-derived stromal/stem (ASCs) cells were fluorescently labeled with a membrane dye and injected into both immunocompetent and immunocompromised mouse strains. Cells were injected either as single cell suspensions, or as self-assembling spheroids. In parallel, cells were purposefully devitalized prior to injection and then implanted in the opposite side in a randomized fashion. These ‘control’ groups were included to determine whether the fluorescent membrane dye would remain localized at the injection site despite the use of nonviable cells. Cell implants and the surrounding tissues were harvested on days 3, 10 and 21 after in vivo delivery and evaluated in a blinded manner. Injection sites were analyzed by fluorescent microscopy, and human cell numbers were quantified using PCR detection of a human-specific endogenous retrovirus (ERV-3). Host response was evaluated by immunofluorescent staining of macrophages.
ERV-3 quantification showed that 95% of the human cells that were viable when they were injected were undetectable at the three-week time-point. Although fluorescent signal persisted for the entire study period, further analysis revealed that much of this signal was located within host macrophages.
These results suggest that human ASCs survive for less than three weeks after injection into even immunocompromised mice, and call into question the notion that human ASCs are immuno-privileged and capable of surviving for extended periods in xenogeneic and/or allogeneic models.
As the promise of cell-based therapies begins to transition to the clinic, a clear understanding of the survival, localization and identity of administered cells over time remains elusive but of great interest. A major limitation relates to various technical challenges associated with the reliable identification and tracking of cells in vivo. Even in the context of preclinical animal models, the sensitive and specific identification of human cells after implantation presents certain challenges. Although a number of methods have been described, most – if not all – should be re-evaluated in the context of recent findings related to cell-derived vesicles, or microparticles [1, 2]. These elements encompass a heterogeneous spectrum of membrane-bound structures ranging in size from 20 nm to 1 μm, released by an ever-expanding list of cell types, and containing DNA, RNA, and cytosolic and/or membrane-associated proteins . The notion that such microvesicles may play a role in the false positive contamination of host cells by labeled donor cells has been reported recently in the literature .
A variety of literature suggests that human adipose-derived stromal/stem cells (ASCs) are immunotolerant/immunoprivileged and can survive for prolonged periods in immunocompromised and even immunocompetent animals [4–7]. In this study we investigated the survival of human ASCs in both immunocompetent and immunocompromised murine models, delivered either as single-cell suspensions or as self-assembled three-dimensional spheroids. The extent of human cell engraftment was quantified over time using PCR detection of an endogenous retrovirus (ERV-3) present in all human cells, but not present in rodent cells. Since the ERV-3 genetic sequence is present as only a single copy per human cell, its detection is directly proportional to the number of human cells present. Using this sensitive and specific method of human cell detection, we demonstrate that nearly 90% of human ASCs that were viable upon injection are undetectable by day 10 after administration into nude mice. Human cell clearance is even faster in immunocompetent animals.
Materials and methods
Isolation and culture expansion of human adipose-derived stromal/stem cells
Human adipose tissue samples were obtained from elective surgical procedures under Institutional Review Board approval at the University of Virginia. We obtained all necessary consent from any patients involved in the study. ASCs were isolated as described previously [8, 9]. Briefly, samples were washed, enzymatically dissociated with Liberase Blendzyme (Roche Applied Science, Indianapolis, USA), and filtered to remove debris . 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 membrane dye (DiI) labeled according to the manufacturer’s protocol (Molecular Probes, Inc., Eugene, OR, USA). Some labeled cells were formed into self-assembling spheroids in hanging drop [11, 12]. Mouse embryonic fibroblast cells (3 T6) were obtained from University of Virginia cell culture core facility and were cultured in Dulbecco’s modified Eagle’s medium/F12 high glucose with 10% fetal bovine serum.
DiI labeling of adipose-derived stromal/stem cells
ASCs were suspended at a density of 1 × 106/ml in culture medium. Five microliters of DiI cell labeling solution were added to the cell suspension, followed by gentle mixing with a pipette. The mixture was incubated at 37°C for 20 minutes. Next, the labeled suspension tubes were centrifuged at 1,500 rpm/200 × g force for 5 minutes at 37°C. This was followed by removal of supernatant and resuspension of the cells in Dulbecco’s modified Eagle’s medium/F12 high glucose with 10% fetal bovine serum at 37°C. This washing procedure was repeated two more times.
Adipose-derived stromal/stem cell implantation into mice
Procedures were performed with approval of the University of Virginia Animal Care and Use Committee. Two strains of mice were used. Thirty-six immunocompetent wildtype (C57BL/6NCr) mice and 36 immunocompromised (Athymic NCr-nu/nu) mice were anaesthetized using ketamine and randomly treated with 300,000 cells either in suspension or preaggregated into spheroids (10 spheroids each comprised of 30,000 cells), followed by appropriate postoperative pain control. Cells delivered as suspensions were injected subcutaneously and into the inguinal region, while cells formulated as three-dimensional spheroids were delivered through an incision into the inguinal fat pad of mice. Implants composed of nonviable cells/spheroids served as parallel controls, implanted in the contralateral side in a randomized, blinded fashion. Nonviable cell implants were generated by overnight incubation at −80°C, thawing at room temperature and confirmed as nonviable with trypan blue dye exclusion and Cell Proliferation Reagent WST-1 (Roche Applied Science).
Harvesting and processing of tissues
Three sets of mice (each set comprising 12 immunocompetent mice and 12 immunocompromised mice) were harvested on days 3, 10 and 21 after implantation. Through random sampling, one-half of the mice from each harvest time point were assigned to be used for histology and one-half of the mice for human cell quantification by PCR detection of ERV-3. The histology specimens were fixed in 10% neutral buffered formalin and were embedded in paraffin while the PCR samples were frozen at −80°C.
Quantification of human adipose-derived stromal/stem cells
Real-time PCR detection of the human/primate-specific ERV-3 was used to evaluate and quantify the presence of human ASCs. Of note, the ERV-3 gene is known to reside at a single locus (on human chromosome 7), enabling a direct correlation between ERV-3 levels and human cell numbers . The primers for the human specific ERV-3 gene were designed as described previously [14, 15]: forward, 5-ATG GGA AGC AAG GGA ACT AAT G; reverse, 5-CCC AGC GAG CAA TAC AGA ATT T (Integrated DNA Technologies, Coralville, Iowa, USA). Preserved samples from injection sites were frozen with liquid nitrogen and ground to powder using a mortar and pestle. DNA extraction was performed with DNAzol (Molecular Research Centre, Cincinnati, Ohio, USA) according to the manufacturer’s protocol. DNA extract from cultured ASCs served as a positive control (that is, ASCs 100%) and DNA extract from an untreated mouse was used as a negative control (that is, ASCs 0%).
Standards were prepared by combining cultured human ASCs and mouse embryonic fibroblast cells (3 T6) in known ratios (ASCs 4.76%, 0.498%, 0.05%, and 0.005%). Accordingly, standards were generated from the mixture of defined numbers (5 × 104, 5 × 103, 5 × 102 and 5 × 10) of human ASCs 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, Hercules, CA, USA). Each reaction was performed using 4.5 μl DNA specimen added to 8 μl PCR reagent mixture comprised of SYBR green and forward and reverse primers. Extracted DNA was assessed for quality and quantity using GeneQuant Pro (Amersham Biosciences, Piscataway, NJ, USA) and each sample was run at 1:10 and 1:50 dilutions in duplicate. The PCR conditions used were: first step, 95°C for 15 minutes; and second step, 45 cycles each with 30 seconds at 95°C (denaturation), 30 seconds at 60°C (annealing) and 30 seconds at 72°C (extension). The 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, with an ERV-3 melting temperature of 87.5°C.
Real-time PCR results
Mouse 3 T6 cells
Human ASC %
CT(n = 6)
Histology and immunohistochemistry
Tissue sections were stained with hematoxylin and eosin, and representative sections were prepared for immunohistochemical staining by deparaffinization with xylene and rehydration through a graded ethanol series. A heat-mediated antigen retrieval technique that included a 20-minute boil in 0.01 M citrate buffer, pH 6.0 (Fisher Scientific, Waltham, MA, USA) was used. After cooling for 1 hour, two separate 5-minute washes in Tris-buffered saline/Tween20 pH 7.4 (Trizma base; Sigma; Tween20; J.T. Baker, St Louis, MO, USA), followed by two 5-minute washes in phosphate-buffered saline were performed. The sections were incubated in 2% horse serum (Sigma, Austin, TX, USA) prepared in phosphate-buffered saline with 0.5% gelatin from cold water fish skin, to inhibit nonspecific binding of the primary antibody. Following incubation in blocking serum, the sections were incubated in primary antibody rat anti-mouse Mac-2 (Cedarlane, Burlington, ON, Canada), dilution 1:1,000, in a humidified chamber for 1 hour. Following this, two 5-minute washes in 0.5% gelatin from cold water fish skin/phosphate-buffered saline were performed. The sections were then incubated in secondary antibody Alexa Fluor 488 donkey anti-rat (Invitrogen, Carlsbad, CA, USA) for 1 hour in a humidified chamber at room temperature. This was followed by three 5-minute washes in phosphate-buffered saline, prior to aqueous mounting (Fisher Scientific).
The immunostained slides were imaged using a confocal Nikon Eclipse TE 2000-E2 microscope (Nikon, Melville, NY, USA) equipped with a 60× Nikon oil immersion objective
The percentage of ERV-3 was obtained with real-time PCR as the fraction of ERV-3 remaining at the implantation site on days 3, 10 and 21. Data were analyzed using SPSS 19.0 (IBM Corp. Released 2010. IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY, USA). Using an independent-samples t test, we compared the percent ERV-3 between the ASCs injected as a cell suspension versus spheroids for different time points (days 3, 10 and 21), separately for the two murine species. We also compared the percent ERV-3 between immunocompetent mice versus immunocompromised mice for the time points studied.
All mice survived the surgical procedure of cell implantation. Out of 72 treated animals, 71 survived to experimental endpoint. One athymic NCr-nu/nu mouse injected with spheroids died on day 7.
PCR for adipose-derived stromal/stem cell presence and quantification
Survival of adipose-derived stromal/stem cells
Day 3 (n = 3)
57.5 ± 3.9
55.6 ± 14.7
0.9 ± 0.8
90.6 ± 8.2
87.6 ± 6.8
0.1 ± 0.03
0.9 ± 0.3
Day 10 (n = 3)
7.1 ± 3.0
11.7 ± 0.8
12.1 ± 1.3
Day 21 (n = 3)
4.8 ± 0.6
6.3 ± 0.9
Histology and immunohistochemistry
A variety of cell-based therapies stand at the threshold of changing the way medicine is practiced. A growing body of evidence now suggests that cell therapies act primarily via paracrine effects [16, 17]. However, questions remain unanswered as to how many cells are needed and how long such cells are needed at a given site in order to effect a reproducible response. To answer these questions, reliable quantitative methods are required that are both specific and sensitive. In other words, one of several important issues related to the future understanding and translation of such therapies to the clinic pertains to quantifying and optimizing cell survival and biodistribution after cell delivery. This challenge remains critical even for pivotal preclinical studies that are necessary to support the safety and efficacy of emerging therapies.
Given the growing interest in exploiting adipose tissue as an abundant cell source, our objective was to evaluate human ASC persistence after implantation into immunocompetent and immunocompromised mice using fluorescent membrane labeling techniques for histological tracking, and PCR detection of human-specific genetic sequences for confirmatory and quantitative analysis. Both sensitive and specific, real-time PCR for ERV-3 detects as few as 50 human cells in 1,000,000 murine cells (0.005%) [14, 15], compared with detection of 5,000 cells/organ reported in green fluorescent protein studies . ERV-3 is present in the human genome as only one copy per cell, on chromosome 7 [13, 14]. As such, ERV-3 can be used to detect and quantify the presence of human cells, and do so without need for cellular manipulation. However, it does not provide histological or morphological data. Our data show that <1% of nonviable ASCs implanted were detectable by PCR detection of ERV-3, 72 hours after implantation. Although detection of ERV-3 does not give a direct confirmation of cell viability, it is conceivable that a close correlation does exist between cell viability and the ability to detect a specific DNA sequence with PCR. It is known, for example, that large DNA fragmentation occurs as early as 5 minutes after the onset of apoptosis. Further breakdown into smaller fragments continues over 2 to 24 hours [19, 20].Based on our PCR data, it appears that human ASCs are cleared quite rapidly in the murine strains used in this study, with clearance in immunocompetent mice occurring faster than in immunocompromised mice. More specifically, nearly 90% of cells that were viable at injection are undetectable by day 10, and only 5% are detectable at the implant site at 21 days. It is possible that a majority of the cells migrate away from the injection site by this early time point. However, the robust fluorescent (DiI) signal that is visible at the implantation sites (Figures 3, 4 and 7) argues strongly against this possibility.
Somewhat surprisingly, human cell clearance also appears to be rapid in the immunocompromised Athymic NCr-nu/nu mouse model, raising concern for suitability of this strain for xenogeneic studies. The rapid undetectability of human cells in vivo is associated with a robust infiltration of host macrophages – even in the athymic mouse. Although mature T cells are missing in athymic mice, the B cells, dendritic cells and granulocytes are all relatively intact, and there is a compensatory increase in macrophages and natural killer cell activity . The co-localization of Mac-2 staining with fluorescent DiI label/signal suggests strongly that ASC implantation stimulates a rapid and robust macrophage infiltrate which correlates with decreasing (human) cell numbers as measured by ERV-3 quantification (Table 2). Indeed, our results show that a significant amount of DiI signal is located within Mac-2 stained macrophages (Figures 5 and 6). The DiI fluorescent signal remained visible throughout the entirety of our study time period; however, it appears that human ASC-related dye transfer and/or persistence can occur in the context of macrophage-mediated phagocytosis of human cells [22, 23], via the exchange of membrane microdomains , and/or via microvesicle and/or exosome [2, 24] transfer. It is possible that the fluorescent DiI stain is itself immunogenic and may accelerate the engulfment of human cells. There could also be tissue-specific and/or species-specific differences in the macrophage response to implanted cells. In this study, we injected human ASCs subcutaneously and into the inguinal fat pad. Adipose tissue is known to contain resident macrophages and can also efficiently recruit additional macrophages when inflammatory stimuli arise [25–27]. It is conceivable that in other tissues the time course or extent of this phenomenon may look different. In addition to tissue-specific responses, it is unclear whether our findings would be similar in other immunocompromised murine models, such as the nonobese diabetic/severe combined immunodeficiency mouse model. Both of these variables deserve further interrogation.
This study also compared the persistence of human ASCs delivered as single-cell suspensions versus three-dimensional spheroids. Contrary to our original hypothesis, this study did not demonstrate any statistical difference in the persistence of ASCs formulated and delivered as three-dimensional spheroids compared with those delivered as cell suspensions. Three-dimensional spheroid cultures are widely believed to better mimic the in vivo condition. They exhibit a higher degree of structural complexity and homeostasis, analogous to tissues and organs, including the presence of self-generated extracellular matrix . For these reasons, we believed that cells delivered as spheroids would succumb less to anoikis, and remain more localized and robust than similar cells delivered as a single-cell suspension. However, the ERV-3 data do not support this hypothesis, perhaps because the macrophage infiltrate is similar regardless of the implant formulation.
Previous studies of implanted cell persistence and migration in vivo
Species of donor and recipient
Method and site of delivery
Cell labeling method
Donor, human ASCs; Recipient, SCID mice
5 × 106cells injected intramyocardially in the peri-infarct region
Transduction with luciferase, GFP
10 weeks: 10% of the human ASCs were localized at the site of injection for 10 weeks. No migration detected. 3.5% differentiated into cardiomyocytes or endothelial cells.
Donor, human HL60 cells; recipient, NOD/SCID mice
Intravenous injection of 20 × 106 cells stained with DIR. Rest of the cell labeling techniques were studied in vitro
Lipophilic dyes: DiI, DiD, DiR, PKH26
2 weeks: lipophilic dyes lead to rapid contamination of neighboring cells. CFSE showed good biocompatibility and staining efficiency and showed little contamination. DDAO was toxic to cells. Quantum dots provided heterogeneous staining that is not suitable for intravital microscopy (IVM). IRDye 800CW had suboptimal excitation by the 633 nm lasers used for IVM in this study
Amine reactive dye: CFSE, DDAO-SE
Nano crystals: quantum dots 70S
Antibodies: IRDye 800CW
Donor, porcine ASCs; recipient, Pigs
Subcutaneous implantation of cells seeded in collagen scaffold
4 weeks: BrdU-labeled ASCs were present but no quantification was done
Donor, MCF7 human breast cancer cells, human cord blood-derived cells, human NeoHep cells, human hepatopancreatic precursors; recipient, NOD/SCID mice
Injection into left lobe of liver, 7.5 × 105 human NeoHep or cord blood cells; tail vein injection, 5 × 106 MCF7 cells; intrapancreatic injection, 5 × 105 hepatopancreatic precursor cells; intracardiac transplantation, 5 × 105 hepatopancreatic precursor cells
DiI and red fluorescent nanoparticles Qdot655
3 weeks: FISH for human-specific Alu sequence and mouse major satellite showed that though many of DiI-labeled cells were human in origin, some were phagocytosed by murine cells. Qdot655 faded during the FISH procedure.
Donor, human ASCs; recipient, BALB/C nu/nu mice
5 × 106cells injected i.m. or i.v.
Transduction with luciferase
75% of cells were lost in first week, the remainder were stable for up to 32 weeks
Donor, sheep MSCs; recipient, Merino-cross sheep
DiI labeling and CFSE
DiI-labeled MSCs showed dye retention for 6 weeks. CFSE showed rapid signal loss over 8 days
Donor, human ASCs; recipient, BALB/C nu/nu mice
106 cells injected i.m., s.c., i.v., i.p. or enclosed in a fibrin matrix
Lipofection and electroporation with luciferase, GFP
3 weeks: cells migrated and accumulated at the ventral side. A higher fibrinogen concentration limited cell mobility in the fibrin matrix
Comparison of contemporary cell tracking methods
Tracking method – mechanism
GFP, luciferase – DNA transfer can be mediated virally (transduction), via liposomes (lipofection), or by electrical parameters (electroporation/transfection)
- Genetic manipulation of cells may alter their function
- Human ASCs with GFP or luciferase resume proliferation normally
- Since they are cytosolic in location; the vector (mRNA/DNA) and/or the protein (GFP) could be transmitted to host cells via fusion elements and/or microvesicle secretion, resulting in contamination. Hence, there is concern for false positivity and their detection may not equate to viability of donor cells
- Detection is sensitive to in vivo non-invasive bioluminescence imaging
- Technique requires serial passages, not suitable for use with fresh uncultured cells
- Many mammalian tissues have endogenous fluorescence
BrdU – this nuclear marker is a thymidine analog that replaces (3H) thymidine and can penetrate cell membranes to incorporate into newly synthesized DNA strands of actively proliferating cells
- Optimal labeling requires longer incubation time
- BrdU labeling has no effects on the ASC differentiation/proliferation and is not cytotoxic
- Not suitable for non-invasive methods of detection
- Does not indicate viability
- Cells lose BrdU rapidly with serial passages
Lipophilic dyes (DiI, DiR) –long-chain carbocyanine dyes with long aliphatic tails that incorporate into the lipid regions of the cell membranes
- Rapidly contaminates neighboring cells by macrophage-mediated phagocytosis, exchange of membrane microdomains, microvesicle and/or exosome transfer
- Easy technique for labeling and identification of cells
- May be cytotoxic to cells
- The dye fades with serial passages
Amine reactive probes (CFSE) – these diffuse into cells and react with cytosolic amine-containing residues to form dye–protein adducts that are retained
- The dye is toxic to ASCs
- Good staining efficiency
- Not suitable for in vivo non-invasive imaging
- Does not correlate with viability
- Contamination of neighboring cells can occur via macrophage-mediated phagocytosis, microvesicle and/or exosome transfer
Nanoparticles – small crystals made up of inorganic molecules; for example, iron oxide, cadmium
- Can be toxic to cells in high concentration and detection is difficult with low concentrations
- Photostable, remain resistant for long periods of time
- Contamination of neighboring cells can occur via phagocytosis, microvesicles and/or exosome transfer
- Can be used for in vivo non-invasive imaging
- Does not correlate with viability
Real-time PCR for endogenous retroviral sequence (ERV-3) – the gene is present as a single copy in the human genome and so can be used to detect the presence of transplanted human cells in animal models
- Not suitable for non-invasive methods of detection
- Gives a quantitative estimate of number of cells
- False positives can occur by macrophage-mediated phagocytosis but this is very low
- Very sensitive and specific
- The gene is already present in the human cells, so there is no need to stain the cells
FISH detection of human-specific cell surface markers or Alu sequences
- Alu sequences occur in large numbers in the primate genome, which makes higher likelihood of a false positive detection by transmission to host cells/macrophages via microvesicles/exosomes
- The gene is already present in the human cells, so there is no need to stain the cells
- Not suitable for non-invasive methods of detection
- Does not correlate with viability
In summary, these studies demonstrate that when human ASCs are implanted into the subcutaneous and inguinal fat tissue of mice, almost all cells are undetectable within 3 weeks using sensitive and specific molecular techniques. In contrast, a robust fluorescent signal from commonly used membrane dyes is readily detectable by microscopy – even from nonviable cell implants. The co-localization of DiI signal with the MAC-2 stain revealed that DiI is taken up by macrophages by phagocytosis and perhaps other mechanisms of cell transfer. Although clearance of ASCs occurred faster in immunocompetent mice compared with immunocompromised mice, there were no significant differences in the persistence of ASCs delivered as cell suspensions versus those implanted as spheroids. In short, with the emerging evidence of microvesicle formation by ASCs and other modes of host cell contamination, we propose that ERV-3 detection by PCR is a useful method for detecting and quantifying the presence of human cells in xenogeneic models. Further studies are needed to help delineate the advantages and limitations of the various methods available for cell labeling, tracking, identification, and quantification.
adipose-derived stromal/stem cell
endogenous retrovirus 3.
The authors would like to thank Mr John M Sanders for helping with immunohistochemistry.
This project was funded in part by the Armed Forces Institute of Regenerative Medicine (AFIRM-1; to AJK) and in part by the National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (to AJK).
- Ogawa R, Tanaka C, Sato M, Nagasaki H, Sugimura K, Okumura K, Nakagawa Y, Aoki N: Adipocyte-derived microvesicles contain RNA that is transported into macrophages and might be secreted into blood circulation. Biochem Biophys Res Commun. 2010, 398: 723-729. 10.1016/j.bbrc.2010.07.008.View ArticlePubMedGoogle Scholar
- Quesenberry PJ, Aliotta JM: Cellular phenotype switching and microvesicles. Adv Drug Deliv Rev. 2010, 62: 1141-1148. 10.1016/j.addr.2010.06.001.PubMed CentralView ArticlePubMedGoogle Scholar
- Lassailly F, Griessinger E, Bonnet D: ‘Microenvironmental contaminations’ induced by fluorescent lipophilic dyes used for noninvasive in vitro and in vivo cell tracking. Blood. 2010, 115: 5347-5354. 10.1182/blood-2009-05-224030.View ArticlePubMedGoogle Scholar
- Bai X, Yan Y, Coleman M, Wu G, Rabinovich B, Seidensticker M, Alt E: Tracking long-term survival of intramyocardially delivered human adipose tissue-derived stem cells using bioluminescence imaging. Mol Imaging Biol. 2011, 13: 633-645. 10.1007/s11307-010-0392-z.View ArticlePubMedGoogle Scholar
- Meyerrose TE, De Ugarte DA, Hofling AA, Herrbrich PE, Cordonnier TD, Shultz LD, Eagon JC, Wirthlin L, Sands MS, Hedrick MA, Nolta JA: In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models. Stem Cells. 2007, 25: 220-227. 10.1634/stemcells.2006-0243.PubMed CentralView ArticlePubMedGoogle Scholar
- Vilalta M, Dégano IR, Bagó J, Gould D, Santos M, García-Arranz M, Ayats R, Fuster C, Chernajovsky Y, García-Olmo D, Rubio N, Blanco J: Biodistribution, long-term survival, and safety of human adipose tissue-derived mesenchymal stem cells transplanted in nude mice by high sensitivity non-invasive bioluminescence imaging. Stem Cells Dev. 2008, 17: 993-1003. 10.1089/scd.2007.0201.View ArticlePubMedGoogle Scholar
- Niemeyer P, Vohrer J, Schmal H, Kasten P, Fellenberg J, Suedkamp NP, Mehlhorn AT: Survival of human mesenchymal stromal cells from bone marrow and adipose tissue after xenogenic transplantation in immunocompetent mice. Cytotherapy. 2008, 10: 784-795. 10.1080/14653240802419302.View ArticlePubMedGoogle Scholar
- Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC: Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells. 2005, 23: 412-423. 10.1634/stemcells.2004-0021.View ArticlePubMedGoogle Scholar
- Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH: Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001, 7: 211-228. 10.1089/107632701300062859.View ArticlePubMedGoogle Scholar
- Parker A, Shang H, Khurgel M, Katz A: Low serum and serum-free culture of multipotential human adipose stem cells. Cytotherapy. 2007, 9: 637-646. 10.1080/14653240701508452.View ArticlePubMedGoogle Scholar
- Amos PJ, Kapur SK, Stapor PC, Shang H, Bekiranov S, Khurgel M, Rodeheaver GT, Peirce SM, Katz AJ: Human adipose-derived stromal cells accelerate diabetic wound healing: impact of cell formulation and delivery. Tissue Eng Part A. 2010, 16: 1595-1606. 10.1089/ten.tea.2009.0616.PubMed CentralView ArticlePubMedGoogle Scholar
- Kapur SK, Wang X, Shang H, Yun S, Li X, Feng G, Khurgel M, Katz AJ: Human adipose stem cells maintain proliferative, synthetic and multipotential properties when suspension cultured as self-assembling spheroids. Biofabrication. 2012, 4: 025004-10.1088/1758-5082/4/2/025004.PubMed CentralView ArticlePubMedGoogle Scholar
- O’Connell C, O’Brien S, Nash WG, Cohen M: ERV3, a full-length human endogenous provirus: chromosomal localization and evolutionary relationships. Virology. 1984, 138: 225-235. 10.1016/0042-6822(84)90347-7.View ArticlePubMedGoogle Scholar
- Lee ST, Chu K, Kim EH, Jung KH, Lee KB, Sinn DI, Kim SU, Kim M, Roh JK: Quantification of human neural stem cell engraftments in rat brains using ERV-3 real-time PCR. J Neurosci Methods. 2006, 157: 225-229. 10.1016/j.jneumeth.2006.04.019.View ArticlePubMedGoogle Scholar
- Yuan CC, Miley W, Waters D: A quantification of human cells using an ERV-3 real time PCR assay. J Virol Methods. 2001, 91: 109-117. 10.1016/S0166-0934(00)00244-5.View ArticlePubMedGoogle Scholar
- Gnecchi M, Zhang Z, Ni A, Dzau VJ: Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008, 103: 1204-1219. 10.1161/CIRCRESAHA.108.176826.PubMed CentralView ArticlePubMedGoogle Scholar
- Suga H, Glotzbach JP, Sorkin M, Longaker MT, Gurtner GC: Paracrine mechanism of angiogenesis in adipose-derived stem cell transplantation. Ann Plast Surg. 2014, 72: 234-241. 10.1097/SAP.0b013e318264fd6a.PubMed CentralView ArticlePubMedGoogle Scholar
- Niyibizi C, Wang S, Mi Z, Robbins PD: The fate of mesenchymal stem cells transplanted into immunocompetent neonatal mice: implications for skeletal gene therapy via stem cells. Mol Ther. 2004, 9: 955-963. 10.1016/j.ymthe.2004.02.022.View ArticlePubMedGoogle Scholar
- Sun XM, Cohen GM: Mg(2+)-dependent cleavage of DNA into kilobase pair fragments is responsible for the initial degradation of DNA in apoptosis. J Biol Chem. 1994, 269: 14857-14860.PubMedGoogle Scholar
- Winter DB, Gearhart PJ, Bohr VA: Homogeneous rate of degradation of nuclear DNA during apoptosis. Nucleic Acids Res. 1998, 26: 4422-4425. 10.1093/nar/26.19.4422.PubMed CentralView ArticlePubMedGoogle Scholar
- Richmond A, Su Y: Mouse xenograft models vs GEM models for human cancer therapeutics. Dis Model Mech. 2008, 1: 78-82. 10.1242/dmm.000976.PubMed CentralView ArticlePubMedGoogle Scholar
- Pawelczyk E, Arbab AS, Chaudhry A, Balakumaran A, Robey PG, Frank JA: In vitro model of bromodeoxyuridine or iron oxide nanoparticle uptake by activated macrophages from labeled stem cells: implications for cellular therapy. Stem Cells. 2008, 26: 1366-1375. 10.1634/stemcells.2007-0707.View ArticlePubMedGoogle Scholar
- Pawelczyk E, Jordan EK, Balakumaran A, Chaudhry A, Gormley N, Smith M, Lewis BK, Childs R, Robey PG, Frank JA: In vivo transfer of intracellular labels from locally implanted bone marrow stromal cells to resident tissue macrophages. PLoS One. 2009, 4: e6712-10.1371/journal.pone.0006712.PubMed CentralView ArticlePubMedGoogle Scholar
- Fevrier B, Raposo G: Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr Opin Cell Biol. 2004, 16: 415-421. 10.1016/j.ceb.2004.06.003.View ArticlePubMedGoogle Scholar
- Cinti S, Mitchell G, Barbatelli G, Murano I, Ceresi E, Faloia E, Wang S, Fortier M, Greenberg AS, Obin MS: Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans. J Lipid Res. 2005, 46: 2347-2355. 10.1194/jlr.M500294-JLR200.View ArticlePubMedGoogle Scholar
- Villena JA, Cousin B, Penicaud L, Casteilla L: Adipose tissues display differential phagocytic and microbicidal activities depending on their localization. Int J Obes Relat Metab Disord. 2001, 25: 1275-1280. 10.1038/sj.ijo.0801680.View ArticlePubMedGoogle Scholar
- Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW: Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003, 112: 1796-1808. 10.1172/JCI200319246.PubMed CentralView ArticlePubMedGoogle Scholar
- Weir C, Morel-Kopp MC, Gill A, Tinworth K, Ladd L, Hunyor SN, Ward C: Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking. Heart Lung Circ. 2008, 17: 395-403. 10.1016/j.hlc.2008.01.006.View ArticlePubMedGoogle Scholar
- Pap E, Pallinger E, Pasztoi M, Falus A: Highlights of a new type of intercellular communication: microvesicle-based information transfer. Inflamm Res. 2009, 58: 1-8.View ArticlePubMedGoogle Scholar
- Lequeux C, Oni G, Mojallal A, Damour O, Brown SA: Adipose derived stem cells: efficiency, toxicity, stability of BrdU labeling and effects on self-renewal and adipose differentiation. Mol Cell Biochem. 2011, 351: 65-75. 10.1007/s11010-011-0712-x.View ArticlePubMedGoogle Scholar
- Schormann W, Hammersen FJ, Brulport M, Hermes M, Bauer A, Rudolph C, Schug M, Lehmann T, Nussler A, Ungefroren H, Hutchinson J, Fändrich F, Petersen J, Wursthorn K, Burda MR, Brüstle O, Krishnamurthi K, von Mach M, Hengstler JG: Tracking of human cells in mice. Histochem Cell Biol. 2008, 130: 329-338. 10.1007/s00418-008-0428-5.View ArticlePubMedGoogle Scholar
- Wolbank S, Peterbauer A, Wassermann E, Hennerbichler S, Voglauer R, van Griensven M, Duba HC, Gabriel C, Redl H: Labelling of human adipose-derived stem cells for non-invasive in vivo cell tracking. Cell Tissue Bank. 2007, 8: 163-177. 10.1007/s10561-006-9027-7.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.