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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Exp Eye Res. 2019 Oct 23;189:107860. doi: 10.1016/j.exer.2019.107860

Fidelity of Long-Term Cryopreserved Adipose-Derived Stem Cells for Differentiation into Cells of Ocular and Other Lineages

Ajay Kumar 1, Yi Xu 1, Enzhi Yang 1, Yiwen Wang 1,2, Yiqin Du 1,3,4,*
PMCID: PMC6938680  NIHMSID: NIHMS1542305  PMID: 31655040

Abstract

Adipose-Derived Stem Cells (ADSCs) have an important contribution in regenerative medicine ranging from testing stem cell therapy for disease treatment in pre-clinical models to clinical trials. For immediate use of stem cells for therapy, there is a requirement of the high dose of stem cells at different time points which can be met by cryopreservation. In this study, we evaluated the characteristics of long-term cryopreserved ADSCs and their regenerative potential after an average of twelve-year cryopreservation. Revived ADSCs were examined for cell viability and proliferation by trypan blue, Calcein/Hoechst and MTT assay. Expression of stem cell markers was examined by flow cytometry, immunostaining and qPCR. Colony forming efficiency and spheroid formation ability were also assessed. Multilineage differentiation potential was evaluated by induction into osteocytes, adipocytes, neural cells, corneal keratocytes and trabecular meshwork (TM) cells. Post-thaw, ADSCs maintained expression of stem cell markers CD90, CD73, CD105, CD166, NOTCH1, STRO-1, ABCG2, OCT4, KLF4. ADSCs retained colony and spheroid forming potential. These cells were able to differentiate into osteocytes, confirmed by Alizarin Red S staining and elevated expression of osteocalcin and osteopontin; into adipocytes by Oil Red O staining and elevated expression of PPARγ2. ADSCs could differentiate into neural cells, stained positive to b-III tubulin, neurofilament, GFAP as well as elevated expression of nestin and neurofilament mRNAs. ADSCs could also give rise to corneal keratocytes expressing keratocan, keratan sulfate, ALDH and collagen V, and to TM cells expressing CHI3L1 and AQP1. Differentiated TM cells responded to dexamethasone treatment with increased Myocilin expression, which could be used as in vitro glaucoma model for further studies. Conditioned medium from ADSCs was found to impart a regenerative effect on primary TM cells. In conclusion, ADSCs maintained their stemness and multipotency after long-term cryopreservation with variability between different donors. This study can have great repercussions in regenerative medicine and pave the way for future clinical trials using cryopreserved ADSCs.

Keywords: Adipose-derived Stem Cells, Cryopreservation, Stemness, Multipotency, Differentiation

Graphical Abstract

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1. Introduction

Human adipose tissue harbors multipotent stem cells which have been utilized for treatment of a number of diseases leading to >100 clinical trials in cell therapy and regenerative medicine (Frese et al., 2016; Gutierrez-Fernandez et al., 2015; http://clinicaltrials.gov, 2016). These cells can be derived readily by liposuction and can be used as an impending source for autologous stem cell therapy. With increasing focus on stem cell therapy in a multifarious array of diseases, the optimization of cell source and freezing conditions has become equally important to compensate for large demands of cells for cell therapy which is often met by upscaling stem cells in laboratory conditions. The ease of harvesting adipose-derived stem cells (ADSCs) put them into hit list for use in Autologous Stem Cell Therapy.

ADSCs have been employed for various ocular disorders. Progranulin (Tsuruma et al., 2014) secreted by ADSCs has been reported to protect photoreceptors from light induced retinal degeneration in animal models. ADSC secreted hepatocyte growth factor has demonstrated a neuroprotective effect in retinal-ischemia perfusion injury model. ADSCs have been induced to differentiate into corneal keratocytes in vitro (Du et al., 2010). ADSCs could facilitate corneal stroma regeneration in a rat wound model (Demirayak et al., 2016). Autologous ADSCs were safe for advanced keratoconus in a clinical trial in Spain (Alio Del Barrio et al., 2017).

With the advent of new technologies, protocols related to cryopreservation and use of stem cells have greatly improved which can pave the way to long term use of these cells. Most of the clinical studies after long-term cryopreservation have been performed on blood stem cells. Previous reports have shown the engraftment potential of bone marrow mesenchymal stem cells (BM-MSCs) and hematopoietic stem cells cryopreserved in liquid nitrogen for a range of 2–11 years with a median duration of 2.8 years. The cells transplanted in different patients were able to engraft even after that prolonged period of cryopreservation (Aird et al., 1992). A similar study reported the engraftment potential of BM-MSCs for 2–7.8 years with a median of 2.7 years and supported the notion of the functionality of these cells in patients with hematological malignancies after a long period of freezing (Attarian et al., 1996). ADSCs resemble to BM-MSCs in terms of their morphology, proliferation and multipotency (Elman et al., 2012, 2014; Tanna and Sachan, 2014). There are very few studies describing the cell viability, proliferation and multilineage differentiation potential of ADSCs after short-term cryopreservation ranging from few weeks to one or two years (Miyagi-Shiohira et al., 2015). In one such study, authors cryopreserved ADSCs for 20 days and described the maintenance of stemness in ADSCs but reduced α4 Integrin expression (Irioda et al., 2016). Yong et. al. cryopreserved ADSCs for three months using reduced concentration of Dimethyl Sulfoxide (DMSO) and reported that ADSCs were able to maintain their viability and stemness (Yong et al., 2015). Shah et. al. demonstrated the reduction in differentiation capacity of ADSCs after 9–10 months of cryopreservation, however, the cells retained the expression of stemness markers (Shah et al., 2016). Another study evaluated the effect of 1–2 years of cryopreservation on ADSCs and found that ADSCs could maintain their proliferative capacity after a transient attenuation and could also differentiate into cells of different lineages (Devitt et al., 2015). A recent report by Shaik et.al. demonstrated that ADSCs maintained their functionality in terms of stem cell marker expression and osteogenic and adipogenic differentiation after a decade long cryopreservation (Shaik et al., 2018). However, this study didn’t explore the capacity of ADSCs to differentiate into neurons or cells of ocular lineage after long-term cryopreservation.

The era of stem cell therapy is largely dependent upon successful cryopreservation of stem cells and most of the basic studies are conducted on freshly isolated cells which are hard to get in high dose to meet the required clinical dose of stem cells for cell therapy. In this study, we report for the first time, the proliferation potential, maintenance of stem cell marker expression and multilineage differentiation potential of human ADSCs maintained in liquid nitrogen for an average of twelve years.

2. Material and Methods

2.1. Cell Cryopreservation and Thawing

ADSCs were isolated and cultured as previously reported (Du et al., 2010; Marra et al., 2011). ADSCs were derived from adipose tissue through liposuction from four donors (aged 38–53 years) with details provided in Supplementary Table S1. Consent was obtained from patients and the study was conducted in accordance with ethical guidelines of University of Pittsburgh. Three samples were cryopreserved for more than twelve years which were also compared with a sample frozen for one year. Approximately 1×106 cells were cryopreserved in one cryovial. Cells were stored in the cryopreservation medium consisting of 70% DMEM/HAMs F12, 20% FBS and 10% DMSO. Cells were frozen in −80°C to let the cells gradually reduce the temperature and were transferred to liquid nitrogen tank 24 hrs later. After long-term storage, these cells were revived by taking out the cryovials out of liquid nitrogen and immediately transferred to a water bath maintained at 37°C. After thaw, cells were centrifuged for five minutes after dilution in culture medium, stained with trypan blue for cell viability, and seeded in T75 culture flasks with medium containing DMEM/HAMs F12 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% Fetal Bovine Serum (FBS, ThermoFisher, Pittsburgh, PA), 100 IU/ml penicillin, 100 μg/ml streptomycin, and maintained in 5% CO2 atmosphere in cell culture incubator at 37°C. The medium was changed every third day.

2.2. Flow Cytometry

The characterization of stemness of thawed cells was first evaluated by stem cell marker expression using flow cytometry. Details of antibodies and their conjugated fluorochromes used for flow cytometry are given in Table S2. Cells were washed and incubated with different antibodies at a dilution of 1:50 or 1:100 for 30 min on ice. Proper compensation and isotype controls were used for different fluorochromes. 5×104 cells per run were acquired by flow cytometer. Unstained cells were used to eliminate background fluorescence. Samples were run on a BD FACSAria (BD Bioscience, San Jose, CA) and analysis was performed using FlowJo_V10 software (FlowJo, Ashland, OR).

2.3. Quantitative Real-Time PCR (qPCR)

ADSCs were grown in defined culture conditions up to 70–80% confluence and were lysed in RLT buffer with gentle scraping. RNeasy Mini Kit (Qiagen, Hilden, Germany) was used for RNA isolation from the lysed cells. 100–500ng of extracted RNAs were used to make cDNAs by using XLAScript cDNA MasterMix (WorldWide Medical Products, Bristol, PA). 20μl of the total SYBRGreen (Applied Biosystems-ABI, Foster City, CA) reaction mix was used to assess gene expression using the qPCR platform StepOne plus 7700 (ABI). The complete list of gene primers used in the study is provided in Table S3. 18S was employed as a housekeeping control and fold change was determined after subtracting the 18S Ct values from genes of interest using formula 2−ΔΔct.

2.4. Trypan Blue Assay

Immediately post-thaw, cells were suspended in culture medium and centrifuged for five minutes at 1500rpm. The pellet was suspended in 1ml culture medium and 10μl of cells were diluted with equal volume of trypan blue. The cell mixture was transferred to the countess cell counting slide and cell number and viability was calculated using an automated cell counter (Countess II FL, ThermoFisher). The percent positive viable and dead cells were calculated by the instrument automatically. Cell viability was measured at least in quadruplicates for each cell type.

2.5. Cell Proliferation and Viability Assay

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay was used for cell proliferation assessment by growing different ADSCs at an initial seeding density of 5×103 per well of 96-well cell culture plates in at least hexapulicates and incubating them in culture medium for 48 hrs. Cells were treated with 10% MTT for four hours. Live cells converted the MTT to formazan crystals which were recovered by lysing the cells with DMSO. The absorbance of purple color formazan crystals was read at 570nm measurement wavelength using 600nm as reference wavelength on Synergy 2 (Biotek, Winooski, VT). Reference wavelength was normalized for each cell type and cell proliferation was calculated with reference to ADSC4.

Live cell staining was done by Hoechst 33342 for nuclei and Calcein red-orange with a dilution rate of 1:500 and 1:1000 respectively. 5×104 cells per well of 12-well plates were incubated with above stains in log phase for ~20 minutes. After that, cells were washed and imaged under a fluorescence microscope (TE200-E Nikon Eclipse).

2.6. Colony Forming Efficiency (CFE) Assay

ADSCs were seeded as single cell suspension at a density of 1×103 cells per well of 10cm2 cell culture dishes. ADSCs were allowed to grow for 7 days to form colonies by individual cell division. The colonies thus formed were stained with a solution of crystal violet prepared at 0.5% in 25% methanol. Colonies were counted using an objective of 2X at Nikon Eclipse TS100 and photographed using EVOS XL core microscope (ThermoFisher). 100% methanol was used for extraction of crystal violet from colonies to quantify its uptake by further reading the absorbance at 570nm and making the comparison by taking ADSC4 as a reference.

2.7. Multilineage Differentiation

Osteogenic differentiation was induced by using osteogenic differentiation kit (ThermoFisher). 1×104 cells were seeded per well of twelve well plates and grown to 70–80% confluence in undifferentiated conditions and then switched to differentiation medium provided in the kit. Cells were maintained in differentiation medium for 21 days by continuous changing media on the third day. For quantification of calcium granule accumulation, cells were stained with Alizarin Red S (Sigma-Aldrich), photographed and then stained cells were incubated with CPC (cetylpyridinium chloride buffer, Sigma-Aldrich) for extraction of the stain. The absorbance of extracted Alizarin Red S was measured at 570nm wavelength on Synergy 2 system. Cells were also lysed in RLT buffer for qPCR to detect expression of osteocalcin and osteopontin. Adipogenic differentiation was induced by plating 1×104 /cm2 of cells and using differentiation kit (ThermoFisher) for sixteen days. Oil Red O (Sigma-Aldrich) binding to lipids was used for identification of differentiated adipocytes which contain oil droplets. EVOS XL core (ThermoFisher) was used to photograph these cells. Cells were also lysed in RLT buffer for qPCR to detect changes of PPARγ2 expression. Neural differentiation was induced by seeding 2×104 cells per well in 12-well plates using a cocktail of growth factors N2 and B27 supplement, fibroblast growth factor/epidermal growth factor (FGF/EGF, 20ng/ml) and 2% KSR (Knockout Serum Replacement) added in Neurobasal medium for the duration of 5–6 weeks. Formation of long axon-like structures was observed. Cells were stained with neurofilament, β-III tubulin and GFAP antibodies and mRNA expression of nestin and neurofilament were detected by qPCR. Keratocyte differentiation was performed using pellet culture as described previously (Du et al., 2005; Du et al., 2010). In brief, 3×105 cells per 15-ml tube were centrifuged forming a pellet and maintained at 37°C/5% CO2. Cells were grown in the culture medium for 2 days followed by changing to the differentiation medium, which is composed of Advanced Minimum Essential Medium (Adv-MEM) supplemented with 0.5mM Ascorbate 2 Phosphate (A-2-P) and 10ng/ml FGF, for 21 days. The medium was replenished every third day without disturbing the pellets. Keratocytes were characterized by immunofluorescent staining with Kera C (recognizing keratocan), J19 (recognizing keratan sulfate) and Collagen V. qPCR was performed to compare keratocan (Kera), ALDH3A1 and Collagen 5a1 mRNA levels. For Trabecular Meshwork (TM) cell differentiation, 2×104 ADSCs were cultured per well of twelve-well culture plate on conditioned extracellular matrix (ECM) derived from primary TM cells and treated with TM conditioned medium in 1:1 ratio with DMEM/HAM’s F12 with 10% FBS. Cells were maintained for two weeks. These cells were passaged further in ECM-free conditions and assessed for expression of various TM cell markers and for response to dex treatment. Passaged cells were cultured in the presence of 100nM dex for seven days. Expression of TM cell markers Chitinase 3-like 1 (CHI3L1) and Aquaporin 1 (AQP1) along with Myocilin (MYOC) was detected in differentiated TM cells by immunofluorescent staining and qPCR.

2.8. Immunofluorescent Staining

After completion of induction, cells were fixed with 2% paraformaldehyde for 15 minutes and permeabilized using Triton X-100 (0.1%) in PBS. The cells were then incubated with blocking buffer composed of 1% BSA (Bovine Serum Albumin) for 30 minutes. Primary antibodies used for neural differentiation included Neurofilament, β-III tubulin and GFAP; for Keratocyte differentiation, Kera C, J19 and Collagen V; for TM cell differentiation, C, AQP1 and Myocilin. OCT4 was stained to confirm the stemness of undifferentiated ADSCs. Fluorescent-conjugated secondary antibodies against Rabbit, Mouse or Goat related to the primary antibodies were used at a dilution of 1:1500 (Table S2). Fluorescent images were captured using an Olympus confocal microscope. The intensity of the fluorescence was compared using ImageJ software (National Institute of Health, Bethesda, MD).

2.9. Conditioned Medium (CM) Preparation

ADSCs were cultured in culture medium DMEM/HAM’s F12 (1:1) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. At 70% confluence, cells were incubated with fresh culture medium devoid of any supplements for 48 hrs, which was harvested from cells after the end of incubation period, centrifuged at 3000rpm for 5 minutes to get rid of any floating cells and used immediately or stored at −80°C for future use.

2.10. Wound Healing Assay

Primary human TM cells were cultured in the DMEM/HAM’s F12 (1:1) supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin, as described previously (Du et al., 2012), to 90–100% confluence. A vertical wound was created using a 100μl micro tip and the medium was changed after the wound to remove any detached cells. 6-well culture plate containing cells was then transferred to live cell time-lapse microscope (Nikon Eclipse Ti) platform by inserting into 6-well inserts, placing on a movable stage which was maintained at 37°C and 5% CO2. Cells were photographed automatically at the same beacons at an interval of 4 hours for 24 hours. At least 6-beacons were assigned per well to capture the dynamics of wound healing in entire plate well in real time. The wound area devoid of cells was measured in pixels using ImageJ. The wound area at 0hr was considered as 100% and the unhealed areas after wound at 12hr and 24hr were measured and compared to the area at 0hr.

2.11. Statistical Analysis

The results presented in this study are mean ± SD. Statistical comparisons were made using oneway ANOVA followed by the Tukey method in statistical package SAS version 9.3. p < 0.05 was considered to be statistically significant.

3. Results

3.1. Cryo-ADSCs Maintained Their Stemness, Viability and Proliferation.

We revived three ADSC strains (ADSC1, ADSC2 and ADSC3) from 3 different donors after an average of 12-year cryopreservation and one ADSC strain from one donor after 2-year of cryopreservation to compare the effects of long-term cryopreservation. The details of primary culture for various ADSCs with donor description is given in Supplementary Table S1. Cell viability was tested immediately after fast thawing by trypan blue assay. We observed that different ADSCs showed different cell viability ranging from 69–92% on average (Fig. S1A). ADSC1 cells at passage 3, from a 53-year old donor, stored for < 14 years showed a medium survival rate (79.4±2.9%). ADSC2 at passage 4, from a 38-year old donor, stored for ~11 years showed the highest survival rate (92.4±1.2%). ADSC3 at passage 3, from a 51-year old donor, stored only 4-month more than ADSC1, had lowest survival rate (69.2±2.7%). ADSC4 at passage 1, from a 49-year old donor, stored for only ~2 year had a lower survival rate (85.2±2%) than ADSC2. After cell attachment, we determined cell viability in adhered cells by Calcein-AM/Hoechst-33342 live cell staining and cell proliferation by MTT assay. The live cell staining showed that maximum cells were viable as shown by uptake of both Calcein-AM and Hoechst-33342 by all cells visible in the field (Fig. S1B). With the same seeding density and culture conditions, these cells had similar proliferation rates and showed no statistically significant difference between different strains of ADSCs as measured by MTT assay (Fig. S1C).

We sought for characterization of stem cell markers CD90, CD166, STRO1, CD73, CD105, NOTCH1, ABCG2, SSEA4 and negative markers CD45 and CD34 which were directly conjugated to different fluorochromes FITC, PE, PE/Cy7, APC, BV510. The antibody information is given in Supplementary Table S2. We observed that all ADSCs were positive to stem cell markers, by flow cytometry, with varied percentage positivity for different markers as shown in Fig. 1A–B. CD90, CD73 and CD105 were found to be significantly higher in ADSC2 as compared to other ADSCs. CD105 was significantly higher in all three long-preserved ADSCs (ADSC1, ADSC2, ADSC3) as compared to ADSC4 while CD90 was lower in ADSC3. CD166 and NOTCH1 were significantly higher in ADSC1 and ADSC2 in comparison to ADSC4 while ADSC3 showed no significant difference from ADSC4. ADSC3 showed a significant higher ABCG2 positivity as compared to ADSC4 while ADSC2 showed significant higher positivity for STRO1 as compared to ADSC4. The expression of pluripotent stem cell marker SSEA4 on all ADSCs was relatively low and there was no significant difference among the ADSCs. The hematopoietic lineage antibodies CD34 and CD45 showed negligible expression in the ADSCs. Immunofluorescent staining of OCT4 showed positive expression in most of the cells in all ADSCs with the least expression in ADSC1 (Fig. 1C). qPCR results showed that all four ADSC populations expressed ABCG2, OCT4 and KLF4 with varied levels as shown in Fig. 1D. Expression of ABCG2 was significantly higher in ADSC2 and ADSC3, while OCT4 was significantly lower in ADSC2. hKLF4 was significantly lower in ADSC1 and ADSC3.

Figure 1. Stem cell marker expression post-thaw.

Figure 1.

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A. Histograms depicting the positive percentage of stem cell markers expressed by various ADSCs B. Various ADSCs showing comparative positive percentage of stem cell markers in bar diagram, C. Immunofluorescent staining of OCT4 in various ADSCs, scale bar-100μm, D. Gene expression analysis by qPCR for various stem cell genes in four different ADSCs. N=3. P value *<0.05, **<0.001, ***<0.0001.

After stemness characterization on both genotypic and phenotypic levels in various ADSCs, we sought to test the CFE in these cells. As shown in Fig. S2A, all four ADSCs were able to multiply and form colonies after seeding as single cells. In comparison, the CFE was significantly lower in ADSC2 (10.8±1.1) as compared to other ADSC populations. ADSC1 showed a significantly lower CFE (21.5±3.7) as compared to ADSC4 (30.9±3.7) but there was no significant difference in CFE between ADSC1 and ADSC3 (26.3±4.9) or between ADSC3 and ADSC4 as shown in Fig. S2B. The extraction of crystal violet showed the significantly higher uptake of crystal violet by colonies formed by ADSC4 (0.92±0.1) as compared to other ADSCs and the colonies formed from ADSC2 (0.49±0.05) and ADSC1 (0.51±0.05) showed significantly lower uptake of crystal violet (Fig. S2C) which further validated the colony counts (Fig. S2B).

Similar to CFE, we also assessed the spheroid forming potential of various ADSCs for 15 days. Since ADSC2 were exhausted for this assay, we performed spheroid formation in ADSC1, ADSC3 and ADSC4 only. As shown in Fig. S3A, all three ADSCs were able to form spheroids and the size of the spheroids increased with time. Quantitative comparison of spheroid size showed significant larger spheroids in ADSC4 till day 7 but no significant difference was observed at day 13 and day 15 between ADSC3 and ADSC4 (Fig. S3B). Spheroid viability analysis by Calcein/Hoechst staining at day 15 showed fully viable spheroids from all ADSCs (Fig. S3C).

3.2. Cryo-ADSCs Maintained the Potential for Multilineage Differentiation.

The multilineage differentiation potential of ADSCs after cryopreservation was tested by inducing them to differentiate into Osteocytes, Adipocytes, Neural cells, Keratocytes and TM cells. Induction of ADSCs to osteogenic lineage after 21 days showed that all ADSCs except ADSC2 were able to differentiate into osteocytes as shown by up taking Alizarin red, a calcium-binding stain (Fig. 2A). The extraction of Alizarin red showed a significant higher uptake of Alizarin red in differentiated cells, as ADSC1 (1.46±0.1), ADSC3 (1.41±0.1) and ADSC4 (1.14±0.16) in comparison to undifferentiated controls ADSC1 (0.08±0.03), ADSC3 (0.05), and ADSC4 (0.08±0.01) respectively while ADSC2 showed no significant difference between differentiated (0.12±0.007) and control (0.07±0.003) cells (Fig. 2B). The comparison between differentiated cells showed no difference between the alizarin red uptake of ADSC1, ADSC3 and ADSC4, but significant higher uptake compared to ADSC2. qPCR analysis for osteogenic gene Osteocalcin showed the highest upregulation of these genes post differentiation in ADSC1 (121.8±13.5 fold) as compared to ADSC2 (1.5±0.1), ADSC3 (1.2±0.04) and ADSC4 (5.1±0.9) (Fig. 2C). Osteopontin expression showed a similar trend with the highest upregulation post differentiation in ADSC1 (59.7±23) compared to ADSC2 (0.1±0.06), ADSC3 (1.12±0.04) and ADSC4 (4.8±4.4) (Fig. 2D).

Figure 2. Differentiation of cryo-ADSCs into Osteocytes.

Figure 2.

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A. Bright field photographs of ADSCs stained with alizarin red before and after osteogenic differentiation, scale bar-50μm, B. Comparative analysis of osteogenic differentiation between various ADSCs by extraction of alizarin red using CPC, C-D. Real-time PCR analysis of osteogenic genes Osteocalcin and Osteopontin respectively in ADSCs pre/post osteogenic differentiation, scale bar-100μm. N=3. P value */ #<0.05, **/ ##<0.001, ***/ ###<0.0001.

Differentiation into adipogenic lineage was assessed by staining the ADSCs with Oil Red O which was taken up by oil droplets accumulated inside cells after adipogenic differentiation. We observed similar results as that of Osteogenic differentiation where ADSC1, ADSC3 and ADSC4 were highly differentiated into adipocytes and ADSC2 could show only a weak staining pattern for uptake of Oil Red O (Fig. 3A). qPCR analysis of adipogenic gene PPARγ2 also showed the highest expression (9.9±0.4) of this gene in differentiated ADSC1 as compared to other stem cells (Fig. 3B).

Figure 3. Differentiation of cryo-ADSCs into Adipocytes.

Figure 3.

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A. Light microscopic images of ADSCs showing cells pre/post adipogenic differentiation, Oil droplets stained red in adipocytes by Oil Red O stain, B. Bar diagram showing qPCR analysis of PPARγ2 in ADSCs, scale bar-100μm. N=3. P value *<0.05, **<0.001, ***<0.0001.

Neural differentiation of cryopreserved ADSCs was evaluated by immunofluorescent staining for neuronal markers β-III-Tubulin, Neurofilament and astrocyte marker glial fibrillary acidic protein (GFAP). The axonal growth, characteristic of neural differentiation was observed in all ADSCs after a defined period of neural induction (5–6 weeks). Confocal microscopy showed that all neural induced ADSCs were positively stained for β-III-Tubulin and Neurofilament which was absent in undifferentiated control cells (Fig. 4A–B). The intensity of antibody staining for β-III-Tubulin appeared less in ADSC1 but that of Neurofilament appeared to be similar in all differentiated ADSCs. Cell number post differentiation was found to be reduced in ADSC1 as compared to other stem cells. Few astrocytes were also observed in the culture after neural differentiation as evident by the positive expression of GFAP for few cells in all four ADSCs (Fig. 4C). The change in neural genes Nestin and Neurofilament was assessed by qPCR. Although all ADSCs expressed Nestin at baseline, there was a significant elevation in the expression of Nestin after Neural differentiation in all ADSCs. The expression of Nestin was increased to 184.8±33.6, 610.1±83, 1597±188.8 and 88.1±6.6 fold in differentiated ADSC1, ADSC2, ADSC3 and ADSC4 respectively as compared to undifferentiated cells (Fig. 4D). Similarly, NF showed a fold expression increase as 859±102.7, 4030±348.8, 659.2±31.6 and 28.2±6.3 for ADSC1, ADSC2, ADSC3 and ADSC4 respectively as compared to undifferentiated controls (Fig. 4E).

Figure 4. Neural differentiation of cryo-ADSCs:

Figure 4.

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A-C. Various ADSCs showing fluorescent staining with neural markers βIII-Tubulin, neuron cytoskeletal marker Neurofilament (NF) and astrocyte marker GFAP respectively on control (ADSCs) and differentiated cells (N-ADSCs), D-E. Bar diagram showing the mRNA expression of Nestin and NF respectively in control and differentiated ADSCs by qPCR, scale bar 50μm. N=3. P value *<0.05, **<0.001, ***<0.0001.

3.3. Cryo-ADSCs Successfully Differentiate to Cells of Ocular Lineage.

We also evaluated the tendency of cryopreserved ADSCs to differentiate into corneal keratocytes. The differentiation into keratocytes was confirmed by wholemount staining on the pellets with Kera C, J19 and Collagen V antibodies. All four ADSC populations showed positive staining with all three antibodies after 21 days of keratocyte differentiation as shown in Fig. 5A. Quantitative comparison of mean fluorescent intensity (MFI) showed significantly higher expression of Kera C, J19 and Collagen V in differentiated ADSC2 as compared to other ADSCs where ADSC3 which displayed least intensity among all for all three antibodies (Fig. 5B–D). qPCR analysis for keratocyte genes keratocan (Kera) (Fig. 5E), ALDH3A1 (Fig. 5F) and Col5a1 (Fig. 5G) displayed upregulated expression after induction in all ADSCs.

Figure 5. Differentiation of cryo-ADSCs into corneal Keratocytes.

Figure 5.

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A. Fluorescence images of corneal keratocyte after wholemount staining with Keratocyte specific antibodies Kera C (Green), J19 (Red), and Collagen V (Magenta) after three weeks of differentiation. DAPI staining nuclei blue, B-D. Bar diagram showing quantification of mean fluorescent intensity (MFI) for three antibodies Kera C, J19 and Collagen V respectively, N=6. E. qPCR analysis for keratocyte marker Keratocan (Kera), F. ALDH3A1 and G. Col5A1 in control and differentiated ADSCs, scale bar-50μm. N=3. P value *<0.05, **<0.001, ***<0.0001.

We also explored the differentiation potential of cryo-ADSCs into TM cells using a novel protocol developed in our laboratory. We carried out this experiment in three ADSCs (ADSC1, ADSC3 and ADSC4) since ADSC2 were exhausted. We observed that all ADSCs could differentiate into TM cells successfully and expressed TM markers CHI3L1 and AQP1 post-differentiation (Fig. 6A). After dex treatment for 7 days, the expression of CHI3L1 and AQP1 reduced. Quantitative MFI comparison of CHI3L1 among various ADSC differentiated TM cells (ADSC-TM) showed a significant increase as compared to undifferentiated controls (Fig. S4A), while no significant difference among differentiated ADSC1-TM, ADSC3-TM & ADSC4-TM (25±4.6, 22.4±4.3 & 22±0.7, respectively, Fig. 6B). The MFI of CHI3L1 was decreased after dex treatment as ADSC1_TM_Dex (18.3±2.2), ADSC3_TM_Dex (20±1.6) & ADSC4_TM_Dex (21.2±1.7, no-significance) (Fig. 6B). qPCR confirmed the intensity analysis results as CHI3L1 was significantly upregulated in ADSC1-TM (172.8±8.3), ADSC3-TM (12.5±0.1) & ADSC4-TM (242.5±15.6) as compared to undifferentiated controls (as reference setting at 1) (Fig. 6C). After dex treatment, CHI3L1 gene was downregulated significantly in ADSC1_TM_Dex (48.2±2.5), ADSC3_TM_Dex (6.3±0.3); however, no decrease was observed in ADSC4_TM_Dex (406.9±20.5). AQP1 intensity was also increased in all ADSC-TM significantly comparing to undifferentiated cells (Fig. S4A), as ADSC1_TM (51.7±1.1), ADSC3_TM (57.3±1.9) & ADSC4_TM (57.9±1.8) while it reduced after dex treatment to ADSC1_TM_Dex (48±2.1), ADSC3_TM_Dex (50.5±2.2) & ADSC4_TM_Dex (55.2±1.5) (Fig. 6D). Myocilin (MYOC) is glaucoma associated gene and its expression generally increases in TM cells after dex treatment, which is one of the characteristics of TM cells (Keller et al., 2018). Immunofluorescence analysis indicated that MYOC was increased after dex treatment in ADSC1-TM (from 21±3.4 to 49.8±3.5), ADSC3-TM (from 26.3± 11.7 to 33.4± 2.7) and ADSC4_TM (from 46.1± 13.5 to 86.8± 24.2) (Fig. 7A–B). qPCR analysis showed a significant increase in MYOC in all ADSC_TM cells after dex treatment, ADSC1_TM (from 7.2±5.4 to 872.2±41.9), ADSC3_TM (from 2.3±0.5 to 152.5±77.7) & ADSC4_TM (from 32.4±13.5 to 476.3±154.7) which further confirmed the TM characteristics of differentiated cells (Fig. 7C). ADSC in undifferentiated state were negative to Myoc as shown in supplementary Fig. S4B.

Figure 6. cryo-ADSCs differentiation into TM cells and responsiveness to dexamethasone (dex) treatment.

Figure 6.

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A. Immunofluorescent images showing AQP1 and CHI3L1 staining on differentiated TM (ADSC-TM) cells from cryo ADSCs and after dex treatment (ADSC-TM-dex), B. Quantification of MFI for CHI3L1, N=14 for ADSC1, N=8 for ADSC3 and N=15 for ADSC4, C. qPCR analysis of CHI3L1 in the ADSCs pre/post dex treatment, N=3, D. Quantification of mean fluorescent intensity (MFI) for AQP1 in TM differentiated cells pre/post dex treatment, N=14 for ADSC1, N=8 for ADSC3 and N=15 for ADSC4, scale bar 200μm. P value *<0.05, **<0.001, ***<0.0001.

Figure 7. Myocilin (MYOC) change and dex responsiveness.

Figure 7.

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A. Myocilin staining on differentiated TM cells with and without dex treatment, B. Bar diagrams showing a quantitative analysis of MFI for MYOC in differentiated TM cells with and without Dex treatment, C. qPCR analysis of MYOC in the ADSCs pre/post dex treatment, N=10, Scale bar- 200μm. P value *<0.05, **<0.001, ***<0.0001.

3.4. Paracrine Regeneration Induced by Cryo-ADSCs.

Lastly, we observed the regenerative effect of complete conditioned media (CM) obtained from cryo-ADSCs on various features of primary TM cells. In TM cell wound healing experiments, we found that CM from cryo-ADSCs was able to promote wound healing. Taking 0 hr wounding area as 100%, CM from ADSC1, ADSC2 and ADSC4 promoted wound healing of TM cells at 12 hours, with wound area reduced to ADSC1_CM (68.1±7.4%), ADSC2_CM (63.6±5.6%) and ADSC4_TM (68.6±4) and at 24 hours to ADSC1_CM (14±11.5%), ADSC2_CM (17.3±11.1%) and ADSC4_TM (26.4±8.3) significantly as compared to controls (83±6.7 & 48.1±23.6 for 12hrs and 24hrs respectively) (Fig. 8A) while ADSC3_CM didn’t show significant reduction in wounded areas (76.4±7.1 & 37.1±11.6 for 12hrs and 24hrs respectively) in comparison to controls. We also evaluated the effect of ADSC_CM on fibrotic marker expression in TM cells after wound. qPCR analysis demonstrated a reduced expression of SPARC (Secreted Protein Acidic and Cysteine Rich), a fibrosis-associated gene (Oh et al., 2013; Rhee et al., 2009; Yun et al., 2014) after treatment with CM from all ADSCs, ADSC1_CM (0.5±0.05), ADSC2_CM (0.6±0.08), ADSC3_CM (0.4±0.05) & ADSC4_CM (0.6±0.03) as compared to the control without ADSC_CM (Fig. 8B) at 24hrs after wound.

Figure 8. Effect of ADSC conditioned media (CM) on TM cell regeneration.

Figure 8.

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A. Bar diagram showing the percentage area of wound healed by CM in comparison to control, N=6, B. qPCR analysis showing the effect of CM on fibrotic marker SPARC. N=6, P value *<0.05, **<0.001, ***<0.0001.

4. Discussion

Our study showed that ADSCs maintained their stemness after an average of twelve-years cryopreservation in terms of stem cell marker expression, colony forming ability and multilineage differentiation potential. The discovery that long-term cryopreserved ADSCs could be induced to differentiate into osteocytes, adipocytes, neurons, keratocytes, and TM cells indicates the potential that long-term cryopreserved ADSCs could be widely used in regenerative medicine in the future. However, not all the ADSCs maintained their stemness to an equal extent. So patient-specific differences should be considered for cell-based therapy.

The cell viability measurements immediately post-thaw indicate that cell death rate is not necessarily related to the storage length. It might be associated with patient individual difference and the process procedures by individuals for freezing and thawing. However, in culture, the viable ADSCs were able to proliferate quickly and showed no significant difference in cell death and proliferation after cell attachment and further culture and passage. The standard guidelines of stem cell characterization outlined the criteria of positive expression of phenotypic markers CD73/90/105 and negative expression of CD34/45 for mesenchymal stem cells (Daley et al., 2016; Kimmelman et al., 2016). In our study, all ADSCs maintained positive expression of stem cell markers after prolonged preservation which is in accordance with the previously reported studies for ADSCs with a short duration of cryostorage. OCT4 is regarded as the governing factor for pluripotency and positive expression of OCT4 by immunofluorescent staining and qPCR confirmed the stemness of cryopreserved ADSCs. It may be worth mentioning that stem cell genes like ABCG2, OCT4 and KLF4 and stem cell CD (cluster of differentiation) markers displayed a varied degree of expression between different markers which were ultimately reflected in the differential potential of stem cells to differentiate into various lineages. However, it may be noted that despite having significantly higher expression of stem cell markers such as OCT4/CD90/CD105/CD73/CD166/STRO1/NOTCH1 at the protein level and ABCG2 and KLF4 at the gene level in ADSC2, the differentiation potential into Osteogenic, Adipogenic and Neural lineage of ADSC2 was lower than other ADSCs. This shows the importance of rigorous testing of ADSCs based on multipotency and not just on the basis of stemness marker expression, for true translation into clinics. Devitt et. al. (Devitt et al., 2015) have reported that cryostorage for more than two years affects the cell viability of ADSCs in comparison to ADSCs stored for <1 year but later this was negated by continued cell growth and ultimately there was no detrimental effect of cryostorage duration on ADSC viability and proliferation. Some reports have also shown the maintenance of cell viability in ADSCs after three months of storage (Yong et al., 2015) and after 2 years of cryopreservation in other types of stem cells where the cryopreservation didn’t alter the viability and expansion potential of stem cells (Kumar et al., 2015). We also observed similar results in our study as there was no significant cell death in any ADSCs obtained from different patients. Previous studies have shown that there was a reduction in cell viability and colony formation ability of ADSCs after cryopreservation. One study compared the ADSC cell viability, CFE before and after 24 days of cryopreservation (Irioda et al., 2016). However, another study by Shah et. al. showed that Colony formation Units (CFU) were not reduced in ADSCs after 9–10 months of cryopreservation (Shah et al., 2016). Our study showed the comparison between different donors where CFE was reduced in case of two strains (ADSC1, ADSC2) while it was maintained in one strain (ADSC3) as compared to the CFE of ADSC4 cryopreserved for short term (2-year). It is worth mentioning that ADSC1 and ADSC3 (both cryopreserved for 13–14 years) didn’t show any difference between their CFE, however, it was reduced comparing ADSC1 to ADSC4, while no difference between ADSC3 and ADSC4. Also, the ADSC2 (passage 4) which were stored for 10 years displayed lowest CFE, even in comparison to cells which were cryopreserved for longer durations. Thus it becomes clear that patient-specific differences, not the duration of cryopreservation play an important role in determining the CFE.

Although derived from adipose tissue, ADSCs have been utilized for bone regeneration (Arjmand et al., 2018; Dai et al., 2016; Tsuji et al., 2014). Previous reports have shown no detrimental effect on the osteogenic potential of ADSCs after two weeks of storage in liquid nitrogen (Liu et al., 2008; Thirumala et al., 2010). In our study, all ADSCs except ADSC2 were able to maintain their osteogenic potential thus providing support for the above findings and confirming that ADSCs can be utilized for osteogenic applications even after twelve years of cryopreservation. However, patient-specific differences might be responsible for the loss of osteogenic potential in ADSC2 over such a long period of cryostorage. Such findings have been reported previously where authors described the absence of alizarin staining in two out of four ADSC cultures after cryopreservation (Shah et al., 2016). Similarly, ADSC2 showed low adipogenic potential while other ADSCs maintained their capacity to differentiate into adipocytes which was in accordance with other studies published before but with short duration of cryopreservation (Arnhold and Wenisch, 2015; Gonda et al., 2008; Miyamoto et al., 2012).

ADSCs have been demonstrated to be useful for treatment of neurodegenerative disorders by differentiation into neural cells like neurons and astrocytes and improving the cognitive and locomotor functions (Castorina et al., 2015; Park et al., 2013). Neurofilament (Kumar et al., 2017a; Kumar et al., 2017b; Van der Gucht et al., 2007) and β-III Tubulin (Menezes and Luskin, 1994) have been reported to be important markers for characterizing neural differentiation in addition to Nestin (Dahlstrand et al., 1995). The maintenance of neural potential in ADSCs as indicated by increased expression of Neurofilament, β-III Tubulin and Nestin post differentiation supports their strong candidature to be used for various neurodegenerative disorders after a long period of cryopreservation.

Corneal keratocytes are main constituents of corneal stroma which get activated to a repair phenotype upon corneal injury (West-Mays and Dwivedi, 2006). Working together with Dr. Funderburgh at University of Pittsburgh, we previously reported the differentiation of ADSCs into keratocytes using ascorbate supplemented serum-free media (Du et al., 2010) and differentiation of keratocytes from Embryonic Stem Cells (ESCs) (Chan et al., 2013; Hertsenberg and Funderburgh, 2016). Stem cells have been previously reported to be employed for congenital corneal diseases (Liu et al., 2010) but differentiation potential of cryopreserved stem cells to corneal keratocytes has not been studied which can provide benefit for long-term use of these stem cells in corneal disorders. In this study, all cryopreserved ADSCs could differentiate to corneal keratocytes as depicted by expression of different keratocyte proteins Collagen V, ALDH, keratocan and keratan sulfate. This offers hope for applying ADSCs in the context of various corneal diseases for corneal regeneration even after cryopreservation.

The TM tissue is important for maintaining the right resistance to drainage of aqueous humor from the eye and maintaining intraocular pressure. Reduced TM cellularity has been reported to be associated with aging as well as glaucoma and increased intraocular pressure in the eye (Alvarado et al., 1984; Alvarado et al., 1981; Duffy and O’Reilly, 2018; Rasmussen and Kaufman, 2014; Vranka et al., 2015). Gonzalez et.al. have demonstrated the presence of progenitor cells in primary human TM cell culture (Gonzalez et al., 2006). We have shown previously that trabecular meshwork stem cells (TMSCs) (Du et al., 2012) can home in TM tissue of the eye and have the capability to differentiate into TM cells (Du et al., 2013) which induces TM regeneration. Other reports have shown the use of iPSC to restore the outflow homeostatic function in open angle glaucoma model of cell loss (Abu-Hassan et al., 2015) and mouse model (Zhu et al., 2016). The TMSCs can specifically home to and repair laser damaged TM tissue (Yun et al., 2018) which confirms the feasibility of cell-based therapy for TM regeneration. CHI3L1 and AQP1 are important proteins of TM cells (Liton et al., 2005; Verkman, 2003). CHI3L1 has been suggested to play a crucial role in the outflow pathway by preventing cell death, protecting against inflammation and by promoting ECM remodeling (Epstein, 2009). AQP1 maintains the outflow facility by controlling cell resting volume (Stamer et al., 2001). The equal tendency of all ADSCs to differentiate into TM cells reflected by the protein expression of CHI3L1 and AQP1 indicates the possibility for use of cryopreserved ADSCs in regenerating TM. Dexamethasone has been reported to induce stiffness in the TM (Yuan et al., 2013) which might lead to development of steroid-induced Glaucoma (Raghunathan et al., 2015). Myocilin is known as a glaucoma associated gene (Tamm, 2002). Myocilin mutations have been reported to be associated with development of glaucoma (Fingert et al., 2002; Jain et al., 2017). Reduction in CHI3L1 and AQP1 accompanied with increased expression of Myocilin in ADSC-TM cells in response to dex treatment reflects the functional responsiveness of differentiated TM cells to steroids similar to TM cells (Keller et al., 2018). This highlight the feasibility of our system in the derivation of functional TM cells from ADSCs.

However, our study had few limitations. We had limited access to ADSCs which restricted the sample number to 4. Future study is needed to evaluate the effect of long-term cryopreservation on a larger scale of ADSCs from different donors and to compare cells from same donors at different passages and different storage periods. Due to limited availability of getting fresh samples, we were also unable to carry out a detailed comparison of the cryopreserved ADSCs with fresh cells. However, we tried to use the cells available with the least cryopreservation time.

Plenty of evidence have shown that stem cells impart their regenerative effect by paracrine factors. The conditioned media obtained from stem cells contain a plethora of growth factors, cytokines, proteins, growth mediators, ECM modulators etc. However, there are very limited reports available on use of paracrine factors of stem cells to promote ocular regeneration. Manguerra-Gagne, et.al. described the first report regarding paracrine factor induced regeneration after stem cell transplantation in laser induced rat glaucoma where they demonstrated the immediate reduction in intraocular pressure after CM injection from hypoxic MSCs (Manuguerra-Gagne et al., 2013). One recent report demonstrated the efficiency of stem cell-extracellular vesicles in promoting neuroprotection and regeneration in glaucoma animal model (Mead et al., 2018). Sparse studies are available on TM regeneration. Our previous report demonstrated the use of corneal stromal stem cells secretome in promoting wound healing and reducing fibrosis in human corneal fibroblasts (Kumar et al., 2018). Demonstration of increased wound healing induced by conditioned medium along with reducing fibrotic marker indicates the feasibility for use of ADSC paracrine factors in TM regeneration which might have implications for glaucoma, but further investigation is needed before establishing it firmly.

Conclusion

In nutshell, this study provides evidence for the functionality of ADSCs after an average of twelve years of cryopreservation. These cells could maintain their viability, colony formation potential along with the ability to differentiate into osteocytes, adipocytes, neurons, keratocytes, and TM cells. ADSCs after long-term cryopreservation thus can be employed for various diseases including bone disorders, neurodegenerative diseases, ocular disorders and also any adipose tissue related abnormalities. However, individual specific differences should be taken into consideration while designing stem cell therapy for patients.

Supplementary Material

1

Highlights:

  • ADSCs were cryopreserved for ~12 years and regenerative potential was observed.

  • Post thaw, ADSCs maintained stemness as well as cell viability.

  • Cryo-ADSCs were able to differentiate into multiple lineage cell types.

  • Cryo-ADSCs gave rise to corneal keratocytes and trabecular meshwork cells.

  • ADSC conditioned media induced trabecular meshwork cell regeneration.

Acknowledgments

The authors would like to acknowledge Dr. Kacey Marra (Department of Plastic Surgery) for providing primary adipose-derived stem cells, Nancy Zurowski (Department of Ophthalmology) for assisting with Flow Cytometry and Kira Lathrop (Department of Ophthalmology) for helping with imaging.

Funding Support

The work was supported by NIH grants EY025643 (YD), P30-EY008098, Research to Prevent Blindness; and Eye and Ear Foundation (Pittsburgh, PA).

Abbreviations

ADSCs

Adipose-Derived Stem Cells

AQP1

Aquaporin 1

BM-MSCs

Bone marrow mesenchymal stem cells

CHI3L1

Chitinase 3-like 1

CFE

Colony Formation Efficiency

CM

Conditioned Medium

ESCs

Embryonic Stem Cells

ECM

Extracellular matrix

DMSO

Dimethyl Sulfoxide

FBS

Fetal Bovine Serum

iPSCs

Induced Pluripotent Stem Cells

MFI

Mean fluorescent intensity

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

TM

Trabecular meshwork

TMSCs

Trabecular meshwork stem cells

ADSC-TM

ADSC differentiated TM

Footnotes

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Disclosure Statement

The authors declare that they have no competing interests.

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