Objectives: Tacrolimus is currently one of the most commonly used immunosuppressant agents to prevent rejection in organ transplant. Although modern immunosuppressive agents have been successful, rejection cannot be completely prevented. Therefore, in organ transplant research, additional treatment methods are being investigated, with the most important one being stem cell therapy. In addition to tacrolimus therapy, stem cell therapy is now clinically applicable, with frequency of concomitant use seeming to expand in the future. In this study, the effects of tacrolimus on stem cells were investigated.

Materials and Methods: Adipose-derived stem cells were treated with tacrolimus at different doses and time points. We analyzed the effects of changes in stem cell proliferation using MTT analysis. Sox2, Oct3/4, and Nanog protein levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure stem cell pluripotency capabilities.

Results: Our observations showed that tacrolimus causes changes in stem cell proliferation and pluripotency, with changes dependent on time and dose.

Conclusions: The dose of tacrolimus and the time of application of stem cells are important in the planning of stem cell therapy in organ transplant.

Key words : Immunosuppression, Rejection, Stem cell pluripotency, Toxicity

Introduction

Improvements in surgical techniques and modern immunosuppressants have resulted in transplant becoming a treatment option for end-stage organ failure, and this is one of the most impressive medical achievements to date. However, it also entails major problems due to immunologic factors. Apart from surgical issues, rejection of the transplanted tissue in the recipient is the principal problem encountered. Modern immunosuppressants have played a major role in preventing these immunologic rejection attacks. Tacrolimus, one such modern immunosuppressant drug, is used more frequently than other immuno­suppressants because it offers a better safety profile with increased long-term patient survival, especially in children and adolescents.¹

Tacrolimus, a prolonged-release drug, inhibits calcineurin, a protein phosphatase required for T-lymphocyte activation.² It thus inhibits rejection attacks by suppressing T-lymphocyte-mediated immune responses. Although the initial dose and maintenance dose could be different, the minimal therapeutic blood concentration level, which is used in the clinic, is 2.4 × 10^-9 M.^3-5 According to transplant type and rejection level, the dose could be increased by 5 times. Concentrations of 10-6 to 10^-7 M of tacrolimus have been reported as high doses.³

Although tacrolimus is better tolerated and associated with fewer episodes of graft rejection than other agents, it does not entirely prevent the immune system from attacking the transplanted organ. Novel treatment methods are therefore being investigated in addition to modern immunosuppression therapies. Stem cell research, a major subject of focus in recent times, is at the forefront of research into new treatments.

Because of their ability to regenerate, differentiate, and especially immunomodulate, stem cells are promising agents for use in transplant and for treatment of various diseases. Although there are stem cells that can originate from many embryonic and adult tissues, the number of studies on mesenchymal stem cells is particularly high because these are easy to harvest and can be obtained at any age. Mesenchymal stem cells exhibit excellent transformation and self-renewal capacities (multiple competence pluripotency) compared with embryonic stem cells.⁶ The hematopoietic-derived stem cell and adipose-derived stem cell (ADSC) mesenchymal stem cell subgroups are the most commonly used in clinical studies. Although pluripotency markers such as Sox2, Oct3/4, and Nanog are highly expressed in both stem cell types, ADSCs are generally preferred in adults because of easier access to adipose tissue and immunologic advantages, such as interleukin (IL)-10 release.⁷

Although numerous clinical studies have observed promising results in mesenchymal stem cell-based treatment, the method and time of application, as well as the stem cell dosage, have been reported to be capable of altering the effects of treatment.^8,9 Investigating the interaction between coadministered drugs, particularly tacrolimus, and stem cells is also important in terms of the health and characteristics of transplanted stem cells. Tacrolimus is known to interact significantly with other drugs.^10-13

No previous studies have investigated interactions between tacrolimus and stem cells. The lack of such studies is particularly significant because these studies could elicit some clues with regard to enhancing the effects of stem cells used for clinical and experimental purposes in organ transplant. Here, we thus investigated the effects of the interactions between stem cells and tacrolimus on stem cell proliferation and pluripotency properties.

Materials and Methods

All animal protocols were approved by our institution’s animal welfare regulatory committee, and all protocols were in conformity with the “Regulation on the Welfare and Protection of Animals Used for Experimental and Other Scientific Objectives” published by the Turkish Ministry of Food and Agriculture in 2011.

Adipose-derived stem cell isolation
Adipose tissue was surgically removed from the inguinal subcutaneous region of Wistar rats weighing from 250 to 300 g. The bilateral inguinal region was shaved after intraperitoneal injection of ketamine/xylazine (50 mg/kg and 10 mg/kg, respectively), and appropriate sterile field isolation was performed. The subcutaneous fat pad was accessed by an incision parallel to the ligament, which was made from a 0.5-cm inferolateral bilateral inguinal ligament. The superficial inferior epigastric artery and vein entering the adipose tissue were coagulated using bipolar cautery. Minimal bleeding occurred during the procedure.

The fat pads (tissue pieces) were collected in phosphate-buffered saline (1× PBS) containing antibiotics (No. P3813; Sigma, St. Louis, MO, USA). All skin incisions were repaired using a simple suture technique with 4-0 silk suture materials. These adipose tissue fragments were incubated at 37°C with 0.075% collagenase type I prepared in 1× PBS. After incubation, pellets obtained by centrifugation were diluted with Dulbecco’s modified Eagle medium (DMEM) (No. 11885-084; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (No. 11885-084; Gibco). The diluted pellet was then passed through a 100-μm filter for mechanical cell separation. Next, the pellet was recentrifuged and dissolved in DMEM (No. 15140122, 1% penicillin-streptomycin; Gibco) and 1% L-glutamine containing 10% FBS. The pellets were incubated at 37°C for 1 week.

Cell culture
Isolated stem cells were incubated with monolayer cultures, including high-glucose supplemented DMEM (4.5 g/L) with L-glutamine (No. 17-905C; Lonza, Basel, Switzerland), nonessential amino acids, sodium pyruvate, 10% FBS, and 1% penicillin-streptomycin-amphotericin B at 5% CO2, 95% humidity, and 37°C.

To investigate how different concentrations of tacrolimus affected stem cells, stem cells were incubated with 1 control (no tacrolimus) and 5 different tacrolimus concentrations (2.4 × 10^-10 M, 2.4 × 10^-9 M, 2.4 × 10^-8 M, 2.4 × 10^-7 M, and 2.4 × 10^-6 M). The control group was established with the administration of physiologic saline solution, which was also used to dilute tacrolimus. The control group was not treated with tacrolimus.

Hematoxylin and eosin staining
Coverslips were covered with poly-L-lysine (No. P8920; Sigma) for 5 minutes and then sterilized with ultraviolet light. The sterile coverslips were next placed in 6-well plates, with ADSCs seeded in the wells. Hematoxylin and eosin staining was applied to show stem cell morphology.

The medium in the wells was removed, and coverslips with attached cells were washed with 1× PBS. The cells were then fixed with 4% para­formaldehyde, and hematoxylin solution was added to the wells, with cells allowed to incubate for 5 minutes. After incubation, the coverslips were washed and allowed to incubate with eosin solution for 2 minutes. The coverslips were then washed again and inverted onto the slide surface and analyzed under a microscope.

Verification of characterization of adipose stem cells by immunofluorescence
Coverslips were covered with poly-L-lysine (No. P8920; Sigma) for 5 minutes and then sterilized with ultraviolet light. The sterile coverslips were then placed in 6-well plates, with ADSCs seeded in the wells. The immunofluorescence method was applied to show stem cell biomarkers.

The medium in the wells was removed, after which coverslips with attached cells were washed with 1× PBS. The cells were then fixed with 4% paraformaldehyde, and blocking solution (1.5 g bovine serum albumin, 0.134 g ammonium chloride, 0375 g glycine, and 50 mL PBS) was added to the wells. Primary antibody (CD29 [sc-9970] from Santa-Cruz, Santa Cruz, CA, USA; CD90 [ab225] from Abcam, Cambridge, MA, USA; CD105 [ab11414] from Abcam; CD45 [ab10558] from Abcam; and CD34 [ab81289] from Abcam) was dropped onto coverslips and incubated at room temperature. Next, secondary antibody (ab150077, ab150117 from Abcam) was dropped onto coverslips. After incu­bation with secondary antibodies, the coverslips were washed and incubated with DAPI (No. H-1200; Vector Laboratories Inc, Burlingame, CA, USA). For the final step, the coverslips were inverted onto the slides and analyzed under a fluorescent microscope.

Proliferation analysis
Adipose-derived stem cells were cultured in 96-well plates at 1000 cells/well. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (No. D8418; Sigma) test was used to measure cell proliferation based on measurement of metabolic activity. To investigate growth rates at different concentrations of tacrolimus, ADSCs were treated with media at 0 M (control), 2.4 × 10^-10 M, 2.4 × 10^-9 M, 2.4 × 10^-8 M, 2.4 × 10^-7 M, and 2.4 × 10^-6 M tacrolimus concentrations. After 24 and 72 hours, media were removed, and ADSCs were incubated in 100 μL of 10% FBS medium with 25 μL MTT solution for 3 hours in the absence of light. At the end of this period, enzyme activity was stopped with dimethyl sulfoxide (No. D8418; Sigma). Measurement at a wavelength of 5540 nm and adipose stem cells growing in media containing tacrolimus (0 M/SF/control) were used as references. Data were analyzed with GraphPad Prism version 7 software (GraphPad Software, Inc., San Diego, CA, USA).

Cell lysate and Western blot analyses
Cell lysates were prepared in lysis buffer containing Triton-X (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 5% glycerol) and protease inhibitor cocktail (No. 4693159001; Merck, Kenilworth, NJ, USA).

The protein concentration of cell lysates was determined using the Bradford assay method (No. 5000006; Bio-Rad Laboratories, Hercules, CA, USA). Proteins were loaded in equal amounts (100 μg), separated using the Western blot method, and transferred to a polyvinylidene difluoride membrane (No. 1620177; Bio-Rad) overnight.

Membranes were labeled with primary antibodies (Nanog [sc-374001], Sox2 [sc-365823], and Oct3/4 [sc-9081], all from Santa Cruz) and horseradish peroxidase-conjugated secondary antibody (5196-2504 and 5178-2504 from Bio-Rad). Clarity-enhanced chemiluminescence Western blot substrate (No. 1705061; Bio-Rad) was used to transfer the labeled protein levels in the membrane to the film.

Statistical analyses
Western blot results were analyzed using Image-J software (Image processing and Analysis in Java; US National Institutes of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/). Graphs and statistical analyses were produced with GraphPad Prism software.

Results

Adipose-derived stem cell isolation and identification of stem cell markers
The morphology of adipose stem cells obtained from adipose tissue was examined with an inverted microscope (Figure 1). To elucidate the morphology, the cells were stained with hematoxylin and eosin, and morphology of stem cells was observed to be consistent with the literature (Figure 2).

CD29, CD90, and CD105 biomarkers should be positive and CD45 and CD34 biomarkers should be negative for an isolated cell to be classified as a stem cell. Immunofluorescence staining of the cells obtained in our study produced CD29-positive, CD90-positive, CD105-positive, CD45-negative, and CD34-negative results (Figure 3A). These results proved that stem cell isolation had been successfully performed (Figure 3). For CD29, CD90, and CD105 antibody, we performed negative control staining; in these experiments, no signals were detected. In addition, we showed CD45-positive and CD34-positive staining for positive control (red signal) (Figure 3B).

Effects of different concentrations of tacrolimus on proliferation of adipose-derived stem cells
To determine whether different concentrations of tacrolimus affected the proliferation of stem cells, the ADSCs were incubated in medium containing different tacrolimus concentrations for both 24 and 72 hours, after which cell proliferation was evaluated using the MTT assay.

As shown in Figure 4, 2.4 × 10^-9 M tacrolimus did not lead to a significant change in stem cell proliferation in the first 24 hours. However, doses of 2.4 × 10^-7 M and 2.4 × 10^-6 M tacrolimus significantly increased the proliferation of stem cells in the first 24 hours. Similarly, proliferation of stem cells at 72 hours increased significantly at these 2 tacrolimus doses (P < .001).

Adipose-derived stem cells were retreated for 3 days with different tacrolimus concentrations. Treatment with 2.4 × 10^-9 M tacrolimus significantly reduced proliferation on the third day (P < .05). In addition, the 2 concentrations observed to increase stem cell proliferation in the first 24 hours also increased it during the first 72 hours (P < .001). No significant change occurred with other tacrolimus concentrations.

Effect of different concentrations of tacrolimus on the pluripotency markers of adipose-derived stem cells
To investigate whether different concentrations of tacrolimus affected the pluripotency characteristics of stem cells, ADSCs were incubated in medium containing 5 different tacrolimus concentrations for 24 and 72 hours, and lysates of the cells were obtained. Oct3/4, Sox2, and Nanog protein levels were determined using Western blot assay. Analyses revealed that the 5 different concentrations of tacrolimus changed the protein levels of the pluripotency biomarkers in the first 24 hours (Figure 5).

Analysis of the change in protein levels revealed significantly lower expression of Oct3/4 in cells treated with 2.4 × 10^-9 M tacrolimus (P < .001). Expression levels of the other pluripotency bio­markers, Sox2 and Nanog, increased significantly. In addition, tacrolimus administered above 2.4 × 10-9 M in the first 24 hours increased pluripotency properties (P < .001).

Another striking and important finding was the significant increase in expression of the Oct3/4 and Sox2 ADSC pluripotency biomarkers in the first 24 hours when stem cells were treated with 2.4 × 10^-10 M tacrolimus (P < .001), with no significant change observed in Nanog expression (P = .7545).

Prolonged tacrolimus treatment of stem cells produced different results. Stem cells treated with 2.4 × 10^-9 M tacrolimus exhibited a significant decrease in all 3 (Oct3/4, Sox2, and Nanog) pluripotency biomarkers, in contrast to the results for the first 24 hours (Figure 6). Similarly, the use of high-dose tacrolimus, in contrast to that shown in the first 24-hour results, reduced the pluripotency properties. Administration of 2.4 × 10^-10 M tacrolimus had less effect on the pluripotency characteristics of stem cells compared with the other concentrations.

Discussion

Tacrolimus is a modern immunosuppressant that has recently improved the success of organ transplant procedures. It inhibits IL-2 mRNA transcription by forming a pentameric complex with intracellular receptor (FKBP12), calcium, calmodulin, and calcineurin. The resulting formation inhibits the effects of the nuclear factor on active T cells. Expression of nuclear factor is required for IL-2 to begin signaling activation of T lymphocytes.¹⁴ However, tacrolimus cannot completely prevent all rejections after transplant. Numerous studies have therefore been performed for the purpose of developing an additional treatment protocol to ensure complete prevention of rejection attacks. In this regard, stem cells, and especially mesenchymal stem cells, have been investigated as an additional treatment.

The pluripotency of mesenchymal stem cells is controlled by transcription factors such as Oct3/4, Sox2, and Nanog.15,16 Protein levels of Oct4 in stem cells have been shown to be directly related to self-renewal capacity.¹⁷ Stem cells lose their ability to regenerate by silencing the Oct4 genes. An ectopic increase in Oct4 levels is known to cause differentiation of stem cells into mesoderm and endoderm cells. Oct3/4 was not only a pluripotency biomarker but also a fate determination feature for stem cells that make up 3 germ layers.

Similarly to Oct3/4, a decrease of Sox2 levels in the stem cell, a member of the Sox family, causes the stem cell to differentiate in the direction of the trophectoderm.18 Unlike other Sox family members, Sox2 protein constitutes a control mechanism on the Oct3/4 protein level.¹⁸

A decrease in Nanog expression in adult stem cells initiates a tendency to differentiation among cells. Nanog is an essential factor in maintaining pluripotency both in vivo and in vitro, but its primary function is to prevent endoderm differentiation.¹⁹

Stem cell research in the field of transplantation is being evaluated under 2 main categories: creation of a 3-dimensional copy of the failed tissue using stem cells and prevention of rejection attacks with immunosuppressive agents.^9,20 Although the production of 3-dimensional organs is promising for the future, the prevention of rejection attacks is currently more critical. Many preclinical and clinical studies have observed that stem cell administration effectively improves organ transplant outcomes and reduces rejection episodes.^8,9,21-24 Adipose-derived stem cells have been reported to be capable of performing an immunomodulatory function in organ transplant, especially in composite tissue transplant, and are regarded as a promising option in the prevention of rejection episodes.^25,26 Similarly to that shown in organ transplant, the use of ADSCs to prevent rejection episodes in allogeneic skin transplant has been reported to yield positive results.²⁷

Because of the wide variety of effects involved, interactions of tacrolimus with other pharmacologic agents may be important. The lack of previous investigations on interactions between ADSCs and tacrolimus, a potential immunomodulator in organ transplant, is a major deficiency in the current literature. We investigated the effects of tacrolimus on ADSCs in terms of dosage and duration. For this, we studied the effects of tacrolimus in the first 24 and 72 hours after administration and with 5 different tacrolimus concentrations.

Our findings showed that stem cells, when treated with 2.4 × 10^-9 M tacrolimus, did not affect proliferation during the first 24 hours. However, proliferation of stem cells decreased when admin­istration of tacrolimus was prolonged (Figure 4). According to our data, the proliferation capacity of stem cells may decrease over time when stem cell transplant/treatment is applied to an individual who is obliged to use tacrolimus for an extended period. The individual’s duration of tacrolimus use should therefore be considered during the planning phase of ADSC therapy.

We found that treatment with 2.4 × 10^-7 M and 2.4 × 10^-6 M tacrolimus affected stem cell proliferation in the first 24 and 72 hours (Figure 4). High doses of tacrolimus (at 2.4 × 10^-7 M and 2.4 × 10^-6 M) have been shown to increase the Th-2-mediated devel­opment of dendritic cells, which are hematopoietic cells.3 In our study, a high tacrolimus concentration likely also increased the proliferation of hemato­poietic stem cells (mesenchymal stem cells), such as ADSCs.

Of interest and importance, our study also showed that tacrolimus at a low concentration (2.4 × 10^-10 M tacrolimus) had a statistically positive effect on the proliferation of ADSCs in the first 24 hours. This may explain why topical forms of tacrolimus, which are used in the short term in composite tissue transplants, are successful as rejection therapy in patients with arm and face transplants. Furthermore, the addition of low-dose tacrolimus therapy to planned stem cell applications facilitates the management of treatment because it will increase the pluripotency of stem cells and improve their success and quality. Although high doses of tacrolimus (2.4 × 10^-7 M and 2.4 × 10^-6 M) resulted in a significant increase in proliferation, it is unlikely that higher doses would be adopted in clinical practice.

When we examined how the pluripotency biomarkers of stem cells varied depending on tacrolimus concentrations, treatment with 2.4 × 10^-9 M tacrolimus in the first 24 hours led to a significant decrease in Oct3/4 protein levels (Figures 5 and 6). When stem cells were treated with 2.4 × 10^-9 M tacrolimus, Sox2 and Nanog protein levels were significantly increased. Sox2 has been reported to exhibit an effect on the Oct3/4 pluripotency biomarker and to be responsible for Oct3/4 protein levels.^28,29 Our findings suggest that stem cells increase Sox2 to compensate for the decrease in Oct3/4 levels, increasing its expression as a result of that decrease. Previous examination of the target binding regions of the transcription factors Oct3/4 and Nanog has revealed that many target genes overlap.³⁰ That study also suggested that Nanog may have an effect on Oct3/4 levels through the Pou5f1 and Sox2 genes, which are target genes. It may be useful to increase the Nanog expression of stem cells as a way to compensate for the decreasing Oct3/4 level and to ensure the continuity of expression of the target genes. Stem cells may have increased expression levels of Nanog and Sox2 in the first 24 hours to maintain the pluripotency properties affected by the reduction in the Oct3/4 protein level. We predicted that high concentrations of tacrolimus would cause a significant increase in stem cell proliferation because these concentrations caused an increase in the 3 pluripotency biomarkers in the first 24 hours.

Examination of the effects of different concen­trations of tacrolimus on pluripotency on the third day revealed a significant decrease in the 3 biomarkers after administration of 2.4 × 10^-9 M tacrolimus compared with the control group without tacrolimus (Figures 5 and 6). Comparisons of data showed that, at 2.4 × 10^-9 M tacrolimus, the change/stabilization of protein levels required to maintain the pluripotency property in the first 24 hours could not be controlled at 72 hours and that pluripotency, one of the most important features of the stem cell, therefore decreased.

Treatment with 2.4 × 10^-7 M and 2.4 × 10^-6 M tacrolimus in the first 72 hours caused a decrease in the pluripotency capacity of stem cells compared with that shown in the control group. However, this decreased capacity had no adverse effects on cell proliferation, with the rate of proliferation actually increasing.

Stem cell therapies have been used in a wide spectrum of different purposes, such as immuno­modulation, rejection therapy, and skin rejuvenation. The present study represents the first investigation of the proliferation and pluripotency ability of stem cells as a result of interaction with different doses and concentrations of tacrolimus. This change is bidirectional, both positive and negative, depending on the tacrolimus concentration and the duration of treatment. These results on the effects of tacrolimus on stem cells should be further elaborated and researched in light of our basic findings, which could lead to improved effectiveness of stem cell use in clinical practice.

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