Comparison of proliferative and immunomodulatory potential of adipose-derived mesenchymal stem cells from young and geriatric cats

Abstract

Objectives

The objective of this study was to compare the ability of adipose-derived mesenchymal stem cells (aMSCs) generated from young vs geriatric cats to proliferate in culture, suppress lymphocyte proliferation and undergo senescence.

Methods

Adipose tissues from five young (<5 years) and six geriatric (>10 years) cats were harvested and cryopreserved for subsequent aMSC isolation and culture. aMSC proliferation in culture was compared via determination of time until passage two and by 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The immunomodulatory capacity of aMSCs was assessed using lymphocyte proliferation assays, and senescence was evaluated using senescence-associated B-galactosidase (SABG) expression. All assays were performed on aMSCs between passage two and passage three.

Results

aMSCs from geriatric cats took significantly longer (P = 0.008) to reach passage two (median 11 days, range 9–22 days) compared with aMSCs from young healthy cats (median 7 days, range 6–8 days). No significant difference was detected between young and geriatric cats in terms of their ability to suppress lymphocyte proliferation. SABG expression was not significantly different between young and geriatric aMSCs.

Conclusions and relevance

Compared with young feline aMSCs, geriatric aMSCs are significantly impaired in their ability to rapidly proliferate to passage two following initial culture, presenting a concern for autologous therapy. Nonetheless, once the cells are expanded, young and geriatric cat aMSCs appear to be equivalent in terms of their ability to functionally suppress T-cell activation and proliferation.

Introduction

The use of adipose-derived mesenchymal stem cells (aMSCs) in veterinary medical therapy has been steadily increasing in both traditional and specialty regenerative medicine practices. MSCs have the potential for therapeutic benefit via their direct regenerative properties, as well as their ability to alter their surrounding environment through their paracrine affects.^1,2 aMSCs have therapeutic potential in cats with a variety of diseases such as kidney disease, asthma, inflammatory bowel disease, gingivostomatitis and arthritis, owing to their anti-inflammatory properties.^3–7 Immunomodulatory effects of MSCs include suppression of lymphocyte proliferation in vitro, decreased production of tumor necrosis factor alpha and interleukin (IL)-6 by stimulated macrophages, decreased production of IL-12 by dendritic cells (DC) and suppression of DCs function.^1,3,8

The use of autologous MSCs for therapy in older animals necessitates harvesting adipose tissue from animals that may be chronically diseased. Recent literature has highlighted the functional deficits exhibited by MSCs collected from aged and diseased donor patients.^9–13 For example, geriatric MSCs from both human and rodent donors have been demonstrated to have decreased proliferation potential, to express increased markers of senescence and to have a decreased capacity for inducing mature angiogenesis,^9–13 as well as poorer therapeutic potential in a mouse model of autoimmune encephalomyelitis.¹⁴ However, little is known regarding the effect of donor age, disease status or collection location on the proliferation potential, characterization and immunomodulatory potential of feline aMSCs. In our experience, aMSCs from geriatric patients have been difficult to grow.¹⁵ This finding suggests that there may be considerable differences in the clinical utility of MSCs derived from certain donor populations, including important implications for autologous vs allogeneic therapy in cats.^16,17 The aim of this study was to compare the ability to proliferate in culture and suppress lymphocyte proliferation of aMSCs generated from young and geriatric cats.

Materials and methods

Adipose tissues from five young (<5 years old) and six geriatric (>10 years old) cats were harvested and cryopreserved for aMSC isolation and culture. The study was approved by the Institutional Animal Care and Use Committee at Colorado State University, and owners signed informed consent forms prior to participation. While cats were undergoing anesthesia for a surgical procedure or dental cleaning, adipose tissue was obtained at a site on the ventral abdomen just caudal to the umbilicus, as previously described.^5,18 For preparation of the adipose tissue for cryopreservation, the tissue was minced and divided into 1 g aliquots in 1 ml freezing medium (11% dimethyl sulfoxide, 14% MSC medium, 75% fetal bovine serum [FBS]), frozen to −80°C using a freezing container (Nalgene Mr, Frosty; Sigma Aldrich) and then stored in liquid nitrogen for no more than 1 year prior to use. Cryopreserved adipose was then later hand-thawed, immediately washed twice with sterile Dulbecco’s phosphate-buffered saline (DPBS; Sigma Aldrich) and then collagenase digested. Approximately 1 g adipose tissue was digested with collagenase (Sigma Aldrich; 1 mg/ml) for 30 mins at 37°C and centrifuged (380 g, 5 mins) to separate the stromal vascular fraction (SVF) from the remaining adipocytes. The SVF was washed in DPBS and plated in MSC medium in tissue-culture flasks. MSC medium consisted of low-glucose Dulbecco’s modified Eagle medium (Invitrogen/Gibco) supplemented with penicillin (Invitrogen/Gibco; 100 U/ml), streptomycin (Invitrogen/Gibco; 100 μg/ml), L-glutamine (Invitrogen/Gibco; 2 mM), 1% essential amino acids without L-glutamine (Invitrogen/Gibco), 1% non-essential amino acids (Invitrogen/Gibco; 1% bicarbonate solution [Invitrogen/Gibco; 7.5%]) and 15% heat-inactivated FBS (Cell Generation). The cells were incubated for 48 h at 37°C and 5.0% CO₂, after which the medium was changed. The remaining plastic-adherent cells were incubated until approximately 70% confluent, with media changes every 2–3 days. The cells were then rinsed with DPBS and detached using 0.25% trypsin-EDTA (Invitrogen/Gibco) for passage to larger flasks to allow for additional expansion.

Comparison of proliferation

aMSC proliferation in culture was measured in two ways: time to passage two (P2), which is a useful marker in the clinical application of MSCs to estimate injection date, and 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Aldrich) assay at passage three (P3). Time to P2 was recorded as the number of days between setting up the culture and P2 when harvest for clinical use would occur. Cells were determined to be ready for passage when confluency reached 70–90%. At the time that cell culture and assays were performed, the disease status of the cats was not known by the individual performing the procedures (LBZ). All subsequent described assays were performed on aMSCs between P2 and P3. Cell survival during culture (an indirect measure of proliferation) was assessed using a MTT assay.¹⁹ For the assay, 5000 MSCs in 100 μl MSC medium were added per well to a 96-well flat-bottom tissue culture plate in triplicate. After 1, 24, 72 and 120 h incubation times, MTT solution was added to each well, and the plates were incubated for 4 h. After 4 h, 0.1 N HCl in isopropanol was added to each well to dissolve the formazan product. Formazan production was quantified by measuring the absorbance on a spec-trophotometer (Synergy HT; Bio-Tek).

Immunomodulatory potential

Inhibition of lymphocyte proliferation assays were performed in pairs using peripheral blood mononuclear cells (PBMCs) harvested from a specific pathogen-free donor cat. PBMCs were separated via Ficoll centrifugation (LSM Lymphocyte Separation Medium; MP Biomedicals), washed with phosphate-buffered saline (PBS), then stained with carboxy-fluorescein N-hydroxysuccinimidyl ester (5 μM; Vibrant CFDA SE Cell Tracer Kit V12883, [Invitrogen]). PBMCs were plated in a 96-well plate at a concentration of 5 × 10⁵ cells/well. MSCs were harvested at P2 and seeded in the 96-well plate at a concentration of 50,000 cells/well. Concanavalin A (ConA; Sigma Aldrich) was added to experimental wells at a concentration of 5 μg/ml. The following experimental groups were used (in triplicate): PBMC control; PBMC and MSC; PBMC and ConA; PBMC, MSC and ConA. Cells were incubated for 96 h at 37°C and 5.0% CO₂, after which time cells were harvested, washed and then re-suspended in FACS buffer (2% FBS, 0.05% sodium azide, 1 × PBS). The amount of lymphocyte proliferation was analyzed via a Cyan ADP flow cytometer (Beckman Coulter). For the purpose of comparison, lymphocytes stimulated with ConA were set to 100% proliferation.

Cell senescence

Analysis for senescence was performed using two different methodologies: manual counting of senescence-associated B-galactosidase (SABG)-positive cells in culture, and Cy5 fluorescence of SABG-positive cells. For both methods cells at P2 were plated into a six-well plate at a density of 100,000 cells/well and incubated for 8 h, to allow cells to adhere. Cells were then fixed and stained using a Cell Signaling Tech B-galactosidase cell senescence kit (Cell Signalling Technology). For manual counting of SABG-positive cells, the aMSCs from five young and five geriatric cats were plated in a six-well plate along with a positive control (passage 14 geriatric aMSCs) and visually assessed for expression of SABG stain. The total number of cells present in the 10× field of view were counted, and the number of SABG-positive cells within this number were identified.

For Cy5 fluorescence of SABG-positive cells images, brightfield and Cy5 fluorescent images were collected to assess cell density and SABG fluorescence,²⁰ respectively, using an Olympus IX3 microscope and a Hamamatsu Orca R2 camera. Each channel was captured using the same exposure settings. Twenty images were taken per sample and quantified in parallel using ImageJ (National Institutes of Health). Cell density was approximated using cell area per field. This was generated by subtracting background, enhancing cellular definition and generating a binary mask to approximate the area of the field covered by cells. Background subtraction was undertaken using the ‘subtract background’ ImageJ function (rolling ball radius 15 pixels). Cellular definition was enhanced through sharpening by applying the ‘unsharp mask’ ImageJ function (radius = 1, mask = 0.60). Outlines were created by generating a cell mask for regions based on brightness and areas greater than 15 square pixels. The outline was then filled to approximate the area covered by cells. SABG was quantified by subtracting a standardized background image from each image then generating an SABG mask for pixels over the threshold value. The standardized background was generated by combining all SABG channels into a single image stack, then applying a minimum intensity projection to create the background image. All masks were verified visually. Mask statistics were then used to calculate percent positive are for each channel in each mask.

Statistical analysis

Proliferation potential was assessed by comparing days to P2 and cell proliferation in the MTT assay at each time point between groups with a Mann–Whitney test using Prism software (GraphPad). Inhibition of lymphocyte proliferation was assessed by comparing the percentage of suppression between groups with a Mann–Whitney test using Prism software. For SABG expression via manual count, the percentage of SABG-positive cells for each cat was identified and the groups were compared with a Mann–Whitney test in Prism software. For Cy5 fluorescence of SABG-positive cells, expression was normalized to cell density for comparison and statistical analysis of percent positive was compared between groups with a Mann–Whitney test in Prism software. For all analyses a P value <0.05 was considered statistically significant.

Results

Cats

All five of the young cats were female domestic shorthairs and were 1.5 years old. The young cats were otherwise healthy with no known comorbidities and were undergoing routine ovariohysterectomy (serum creatinine 0.9, 0.9, 1.1, 1.1 and 1.1 mg/dl, respectively). In the geriatric group there were two spayed females and four castrated male cats, and breeds included domestic shorthair (n = 2), domestic longhair (n = 2) and Siamese/Siamese mix (n = 2). Average age was 14.1 years (range 10–19 years). In the geriatric group, three cats from which samples were collected had previously been diagnosed with chronic kidney disease (serum creatinine 1.8, 2.8 and 11.0 mg/dl, respectively) and three cats had neoplasia (serum creatinine 0.9, 1.0 and 1.6 mg/dl, respectively).

Comparison of proliferation

aMSCs from geriatric cats took significantly longer (P = 0.008) to reach P2 (median 11 days, range 9–22 days) in culture compared with aMSCs from young cats (median 7 days, range 6–8 days) (Figure 1). When all cats with serum creatinine >2.0 mg/dl were excluded from analysis, aMSCs from geriatric cats still took significantly longer (P = 0.02) to reach P2 (median 10.5 days, range 9–11 days) compared with aMSCs from young healthy cats (median 7 days, range 6–8 days). MTT assay performed in pairs at P3 indicated no significant difference in proliferation ability between the two groups at 1, 24, 72 and 120 h incubation time points when measured via colorimetric assay (Figure 2). One geriatric cat (22 days to P2) could not be expanded beyond P2 on two separate attempts and therefore could not be used in the other assays.

Figure 1

Proliferation time to passage 2 (P2). Adipose-derived mesenchymal stem cells (aMSCs) cultured from geriatric cats took significantly longer (P = 0.008) to reach P2 (median 11 days, range 9–22 days) compared with aMSCs from young healthy cats (median 7 days, range 6–8 days). Cats with serum creatinine >2.0 mg/dl are depicted by triangles

Figure 2

MTT proliferation assay. Proliferation was measured by MTT assay in triplicate at baseline (1 h), 24, 72 and 120 h in culture. No significant difference was seen between the two populations at any time point

Immunomodulatory potential

When the ability to suppress lymphocyte proliferation was compared between young (median 58.5%, range 43.8–72.4%) and geriatric (median 47.5%, range 37.4–78.6%) aMSCs, no significant difference was observed between the two groups (Figure 3).

Figure 3

Suppression of lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from a donor cat were stimulated with concanavalin A (ConA), then incubated with adipose-derived mesenchymal stem cells (aMSCs) from young or geriatric cats in triplicate. No difference in suppression of lymphocyte proliferation was seen between the two populations

Cell senescence

When expression of SABG staining was assessed in young vs geriatric aMSCs, no significant difference in SABG expression was seen between young (median 0.72% SABG-positive cells, range 0–2.1% SABG-positive cells) and geriatric (median 0.71% SABG-positive cells, range 0.51–4.20% SABG-positive cells) cats using a manual count assessment in comparison with positive control (median 10.3%, range 1.9–77.5% SABG-positive cells). A significant difference was also not seen in SABG expression seen between young (median 13.7% SABG-positive, range 13.1–14.3% SABG-positive) and geriatric (18.5% SABG-positive, range 17.4–19.6% SABG-positive) cats when the Cy5 fluorescence methodology was used (Figure 4).

Figure 4

Senescence-associated B-galactosidase (SABG) expression. When the number of SABG-positive cells was assessed using Cy5 fluorescence, no significant difference in SABG expression was observed between young (n = 2) and geriatric adipose-derived mesenchymal stem cells (n = 2). Twenty replicates for each cat are shown, to demonstrate consistency of results

Discussion

The aim of this study was to explore the possible effects of donor age on the functional properties of feline aMSCs. Thus, aMSCs from young and geriatric cats were compared in terms of their proliferation potential in culture, ability to suppress lymphocyte proliferation and degree of cellular senescence in culture. aMSCs from geriatric cats took significantly longer (P = 0.008) to reach P2 (median 11 days, range 9–22 days) compared with aMSCs from young, healthy cats (median 7 days, range 6–8 days). Although subjective, we find time to P2 to be valuable for the clinical application of MSCs as it is important for predicting when cells will be ready for injection. Subjectively, it was noted that the two geriatric cats with the longest time to P2 both had serum creatinine >2 mg/dl. When these cats were excluded from comparison of time to P2 between groups, there was still a significant difference between young and geriatric cats. The delay in the initial expansion phase of MSC growth in geriatric aMSCs could not be attributed to increased senescence of geriatric aMSCs, as determined by SABG expression. Despite differences in age and presence of azotemia and neoplasia in some geriatric cats, no significant difference was found between the two age groups in the ability of aMSCs to suppress lymphocyte proliferation.

The finding that geriatric cats were impaired in their early proliferative potential, as determined by time to P2, is consistent with what we have reported previously when attempting to culture aMSCs from older cats.¹⁵ This phenomenon may significantly limit the use of aMSCs for autologous therapy, as these geriatric cells will take longer to expand to a clinically useful cell number (which is achieved at P2 with our laboratory’s current protocol for young, normal cats) and may stop growing entirely during the attempt, as seen with adipose from one of the geriatric cats in this study. It should be noted that cells that are growing poorly may take on a more flattened morphology, which might influence the interpretation of the confluency of the culture. Additionally, there is some variability inherent to determining confluency of a flask. In our laboratory, we utilize the recommended 80% confluency to determine time of passage but acknowledge potential variability of ± 10% given that cells are often not similarly confluent throughout the flask. In the present study, percent confluency was also necessarily variable between 70% and 90%, in order to harvest cultures in pairs for further assays and prevent increased confluency that can lead to contact inhibition. A less subjective characterization of the differences between young and geriatric cells would be gained by assessing MSC counts within SVF and characterizing cellular morphology and confluency more objectively. Lower numbers of aMSCs in the SVF isolated from the adipose tissue is one possible mechanism that would explain the decreased initial proliferation in geriatric aMSCs as it has been shown that in humans and dogs the overall quantity of cells in SVF decreases significantly with age.^21,22 To date, there are no specific markers for feline aMSCs and therefore the stem-cell portion of the SVF could not be accurately assessed. Additionally, owing to limited amounts of adipose tissue available, total viable cell numbers per gram of adipose tissue or population doubling could not be evaluated, and these are significant limitations of the current study. However, we feel that, despite these limitations, the blinded assessment of time to P2 still has value for the clinical application of feline aMSCs.

Difficulty with expanding geriatric cells significantly limited the type and number of assays that could be performed in the present study. It also potentially biased the results of the present study, as one geriatric cat could not be expanded beyond P2 on two separate attempts and therefore could not be used in subsequent assays. Similar difficulties with proliferation of cells from older donor populations have also been found in humans, with significant decreases in proliferation reported for donors aged 40–68 years ^10–13 Further investigation regarding the effects of age on proliferation are necessary to gain a better understanding of the mechanism behind this effect in order to overcome this hurdle in cellular therapy.

One possible mechanism for an age effect on proliferation is cellular senescence. Several studies in humans have documented increased cellular senescence in MSCs obtained from older donors,^11–13,23 although not in all studies was SABG staining found to be significantly different between age groups.¹² In the present study we did not find that the reduced proliferative potential seen in initial culture of aMSCs from geriatric cats was related to increased cell senescence. However, these assays were limited by cell availability, and additional studies with alternative methods to assess senescence would be needed to demonstrate definitively that senescence is not an influencing factor on decreased proliferation in geriatric feline aMSC cultures.

Potential differences in immunomodulatory potential and proliferative potential of aMSCs derived from diseased vs healthy cats have not been previously investigated. As this diseased population is the target group for autologous stem-cell therapy treatment, it is essential to determine whether similar beneficial effects are achieved when compared with allogeneic aMSCs from young, healthy donors. In the current study, all six geriatric cats were diagnosed with either neoplasia or chronic kidney disease, and all of the young cats were healthy. Although the inclusion of azotemic cats introduced additional variability, it also alluded to another factor that may affect proliferation potential. Subjectively, it was noted that geriatric cats with serum creatinine >2 mg/dl had extended growth time in initial culture. In order to evaluate variability introduced by significantly uremic cats, and assess both age and uremia as variables, statistical analysis was performed both with and without these cats. Interestingly, the negative effect of increased age on proliferation potential remained even when cats with serum creatinine >2 mg/dl were not included in the analysis. Previous studies have demonstrated that MSCs obtained from uremic rats have reduced proliferation in culture, premature senescence and decreased capacity to induce angiogenesis.^24–26 Observations have been mixed as to whether this affects both aMSCs and bone marrow-derived MSCs (bMSCs), and at least one study demonstrated that aMSCs are not as susceptible to uremic effects as bMSCs.²⁷ Although sample size was a limitation in the current studies and definite assessment of the effects of uremia cannot be drawn, the preliminary finding of decreased proliferation potential in some uremic individuals merits additional future study.

Three of the geriatric cats utilized in the study had some form of neoplasia and it is possible that the presence of neoplasia could have an effect on MSC proliferation. Extensive research is currently ongoing regarding the many ways that MSCs and neoplastic cells interact. A recent study assessed stromal cultures from patients with prostate cancer in comparison with bMSCs from healthy donors and cell cultures from normal prostate tissue, and no difference in growth kinetics was observed.²⁸ However, studies have shown an effect of oncoproteins on MSC senescence, as demonstrated by increased SABG expression, but this was not noted in the current study.²⁹ Additional study is warranted to better assess the effects of age and disease on proliferative and immunomodulatory potential for feline aMSCs. Indeed, the original design of this study was to assess a group of healthy geriatric cats; however, no cats could be identified for sample collection.

Feline foamy virus infection has recently been shown to negatively affect feline autologous aMSC proliferation and expansion in culture.⁷ The adipose tissues collected for this study were not tested for feline foamy virus as this was not a known factor at the time the study was completed; however, no multi-nucleated foamy cells were documented in our cell cultures. If feline foamy virus is more prevalent in the geriatric cat population it could be a contributing factor to the decreased proliferation potential observed in the current study. Additional studies are necessary to determine the prevalence and implications of this phenomenon for feline MSC culture.

Some studies have indicated that aMSCs may not be as affected by donor age as bMSCs, particularly when a certain medium is used. In one previous study in humans, when keratinocyte serum-free medium (KSFM) was utilized for expansion of aMSCs no effect of donor age was seen on proliferation potential, telomere length or differentiation capacity of aMSCs from individuals as old as 60 years.³⁰ In another study, when aMSCs were compared with bMSCs from the same individual, geriatric aMSCs displayed less cellular senescence, greater proliferation potential and osteogenic differentiation capacity than bMSCs.³¹ The latter study also used KSFM media and these results imply that further optimization of culture conditions may be able to overcome inherent differences in aMSC proliferation potential.

Although geriatric aMSCs were significantly slower to reach P2, once they reached this passage their ability to suppress lymphocyte proliferation was indistinguishable from that of aMSCs from younger patients. The effects of donor age on immunomodulatory function have only been evaluated in a limited number of studies. However, in one study which introduced the concept of ‘immunosenescence’ with donor age, aMSCs from geriatric rats failed to suppress CD4 T-cell proliferation, whereas the aged MSCs did suppress CD8 T-cell proliferation with the age of the donor.⁹ This study also found that young rat bMSCs exerted more significant inhibition of lymphocyte proliferation for both T-cell populations than geriatric rat bMSCs.⁹ In the current feline study the effect of donor age on the ability to inhibit lymphocyte proliferation in specific populations of T cells was not assessed.

Conclusions

The results of this study demonstrate that geriatric feline aMSCs are impaired in their ability to rapidly proliferate to P2 following initial culture, but appear to have similar ability to suppress mitogen-stimulated lymphocyte proliferation in vitro when compared with aMSCs from young cats. Additionally, uremia may affect proliferation potential and this influence should be further investigated. This study has important implications for the use of autologous aMSCs in therapeutic applications in the aging cat population; although the efficacy of these cells does not appear to decrease with donor age, the ability to produce enough cells in culture in a timely manner for clinical use may be inadequate in geriatric patients. Additional work is warranted to determine the mechanism of this deficiency in proliferation potential of geriatric MSCs so that the efficiency of expanding autologous feline MSCs in culture can be improved.

Footnotes

Acknowledgements

The authors gratefully acknowledge the surgeons who participated in collection of adipose tissue, and Dr Ann Hess for statistical consultation.

Accepted: 1 November 2016

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study received financial support from a Colorado State University Center for Companion Animal Studies Young Investigator Grant, Frankie’s Fund for Feline Stem Cell Therapy, and the Charles R Shipley Foundation.

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