Regenerative therapy involving stem cell transplantation has become an option for the radical treatment of diabetes mellitus. Disruption in the clock genes of stem cells affects the homeostasis of transplanted tissues. This is possibly the first study to examine the circadian rhythm of genes in transplanted adipose-derived mesenchymal stem cells derived from a patient with type 2 diabetes mellitus (T2DM-ADSC). The clock genes (PER2, CLOCK1, CRY1, and ARNTL[BMAL1]) exhibited similar daily fluctuations in phase and amplitude between a group transplanted with adipose-derived mesenchymal stem cells derived from a healthy individual (N-ADSC) and a group transplanted with T2DM-ADSC. The findings demonstrated that clock genes in stem cells are synchronized with those in living organisms. Moreover, mitochondrial genes and functions showed daily fluctuations in the N-ADSC group. However, such fluctuations were not noted in the T2DM-ADSC group. In the T2DM-ADSC group, daily fluctuations in MTATP8P1 and NDUFA7_2 genes disappeared. In the N-ADSC transplant, the results showed the circadian rhythm in the number of mitochondrial DNA copies, Mitophagy, the mitochondrial membrane potential and NF-kB signaling. In contrast, no daily fluctuations were observed in the T2DM-ADSC transplant. The circadian rhythms of mitochondrial function and NF-kB signaling revealed in this study may be a new marker for the efficiency of stem cell transplantation in patients with diabetes.
Article
The circadian rhythms of mitochondrial function after transplantation of mesenchymal stem cells
https://doi.org/10.21203/rs.3.rs-3108285/v1
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Diabetes mellitus is a disease characterized by long-term abnormalities in sugar metabolism due to decreased secretion of insulin and increased insulin resistance1,2. According to the 9th Edition of the International Diabetes Federation Diabetes Atlas, in 2019, there were 463 million patients with diabetes worldwide. As the disease progresses, complications of diabetic retinopathy (e.g., sight loss and renal failure) begin to occur3,4. Improvement in lifestyle habits and insulin replacement therapy are typical measures utilized for the treatment of diabetes5. In recent years, regenerative therapy using stem cell transplantation has become an option for radical treatment in this setting6–8.
Regenerative therapy for diabetes involves the use of various stem cells9. For example, mesenchymal stem cells (MSCs), which can be stably supplied, are transplanted in an attempt to promote the secretion of proliferative and tissue regeneration factors10. MSCs can be obtained from diverse tissues11,12. Tissues contain higher numbers of adipose-derived mesenchymal stem cells (ADSCs) than bone marrow-derived MSCs13. Reports indicated that ADSCs produce large quantities of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF)14. Accordingly, ADSCs have been attracting attention as excellent cell material for regenerative therapy in patients with rapidly progressing severe diabetes15. Efficiency is the key to effective regenerative therapy by ADSC transplantation. However, it remains unclear whether autologous ADSCs derived from patients with underlying diabetes can achieve similar engraftment to that of ADSCs extracted from healthy individuals.
We previously evaluated the N-ADSC (ADSCs derived from a healthy individual), T1DM-ADSC (ADSCs derived from a patient with type 1 diabetes mellitus [T1DM]) and T2DM-ADSC (ADSCs derived from a patient with type 2 diabetes mellitus [T2DM]) by using transmission electron microscopy and next-generation sequencing16–19. The results revealed that the nuclei of T2DM-ADSC were hypertrophied, and the crista structure of the mitochondria was denatured16. In T2DM-ADSC, the expression of Emerin (a protein composing the nuclear membrane) was low, and the activity of mitochondrial enzymes was reduced16. We also demonstrated that the transplantation efficiency of T2DM-ADSC was lower than that of N-ADSC19. Subsequently, we performed an exhaustive mRNA analysis using next-generation sequencing and identified glucose-6-phosphatase catalytic subunit 3 (G6PC3) and insulin-like growth factor 1 (IGF1) as responsible genes for the difference in transplantation efficiency19. Moreover, it was revealed that the reduced transplantation efficiency of T2DM-ADSC was improved by knocking down G6PC3 and IGF119. These results have clarified the characteristics of T2DM-ADSC. However, post-transplantation functional evaluation has not been previously conducted.
Previously reported clock genes include the period circadian regulator (PER), clock (CLOCK), and cryptochrome (CRY)20. These genes are indispensable for maintaining homeostasis20. Clock genes exist in most cells in the human body, and the central clock controlling these genes is situated in the suprachiasmatic nucleus (SCN)21. In preceding studies, we analyzed the functions of clock genes using models of diabetes and obesity22,23. Some studies reported that long-term disruption in the phase and amplitude of clock genes in stem cells upsets tissue homeostasis24–26. Therefore, it is important to monitor the circadian rhythms of clock genes and genes regulated by clock genes after the transplantation of stem cells.
The purpose of this study was to clarify the circadian rhythms of genes after the transplantation of stem cells derived from a patient with T2DM.
N-ADSC and T2DM-ADSC exhibited similar circadian rhythms of the clock genes in terms of phase and amplitude after transplantation
The results of next-generation sequencing revealed that the N-ADSC transplants exhibited daily fluctuations in the clock genes PER1, PER2, PER3, CRY1, CRY2, and aryl hydrocarbon receptor nuclear translocator like (ARNTL) (basic helix-loop-helix ARNT like 1 [BMAL1]). In contrast, daily fluctuations were not noted in the clock genes CLOCK1, CLOCK2, CLOCK3, and CLOCK4 (Fig. 1a,b). Similarly, the T2DM-ADSC-transplants showed daily fluctuations in PER1, PER2, PER3, CRY1, CRY2, and ARNTL (BMAL1), whereas such fluctuations were not observed in CLOCK1, CLOCK2, CLOCK3, and CLOCK4 (Fig. 1c). In the Z-score display of next-generation sequencing, daily fluctuations were noted in PER1, PER2, PER3, CRY1, CRY2, and ARNTL (BMAL1) in both the N-ADSC (Fig. 1c) and T2DM-ADSC transplants (Fig. 1d). Next, quantitative PCR was performed to analyze daily fluctuations in the clock genes PER2, CLOCK1, CRY1, and ARNTL (BMAL1) (Fig. 1e–h). The clock genes PER2, CLOCK1, CRY1, and ARNTL (BMAL1) exhibited similar daily fluctuations in terms of phase and amplitude in both transplants (Fig. 1e-h). Significant differences in expression were observed only in CLOCK1 at ZT10 and CRY1 at ZT6. At ZT6, the expression of CRY1 was significantly increased in the T2DM-ADSC transplant compared with the N-ADSC transplant (Fig. 1g). At ZT10, the expression of CLOCK1 was significantly decreased in the T2DM-ADSC transplant compared with the N-ADSC transplant (Fig. 1f).
The expression of mitochondrially encoded ATP synthase 8 pseudogene 1 ( MTATP8P1 ) and NADH:ubiquinone oxidoreductase subunit A7_2 ( NDUFA7_2 ) varied between the light and dark periods
Between the N-ADSC and T2DM-ADSC transplants, next-generation sequencing detected great variations in the following categories related to tissue regeneration: cell morphology; embryonic development; hair and skin development and function; organ development; organismal development; and tissue development (Table 1). Based on next-generation sequencing, the genes that exhibited variation in the expression between the N-ADSC and T2DM-ADSC transplants were categorized (Fig. 2a). Genes with considerably decreased expression in the T2DM-ADSC transplant were extracted as 17 candidate genes (Fig. 1b). The results were performed for these 17 genes to analyze their expression levels at two time points (ZT6 and ZT18 in the light and dark period, respectively). In the N-ADSC transplant, two genes (MTATP8P1 and NDUFA7_2) showed significantly lower expression levels at ZT18 (dark period) versus ZT6 (light period). In contrast, significant variations were not recorded in the T2DM transplant (Fig. 2c, q). The remaining 15 genes did not show any significant variations in the expression between the two aforementioned time points (Fig. 2d–p, r, s). The MTATP8P1 and NDUFA7_2 genes that showed variations between the two time points were related to mitochondria. Thereafter, we focused on the daily fluctuations in mitochondrial genes.
Between N-ADSC and T2DM-ADSCs transplants, daily fluctuations in mitochondrial genes showed variations in phase
Fifteen highly expressed mitochondrial genes were identified, namely mitochondrially encoded ATP synthase membrane subunit 6 (MT-ATP6), MT-ATP8, mitochondrially encoded cytochrome c oxidase I (MT-CO1), MT-CO2, MT-CO3, mitochondrially encoded cytochrome b (MT-CYB), MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, mitochondrially encoded 12S rRNA (MT-RNR1), and mitochondrially encoded 16S rRNA (MT-RNR2) (Fig. 3). In the N-ADSC transplant, the mitochondrial genes MT-ATP6, MT-ATP8, MT-CO2, MT-ND1, MT-ND3, and MT-ND4L showed daily fluctuations, reaching their lowest levels at ZT10 and ZT22 (Fig. 3a, b). Moreover, daily fluctuations in the mitochondrial genes MT-ATP6, MT-ATP8, MT-CO2, MT-ND1, MT-ND3, MT-ND4L, and MT-ND5 differed in phase between the T2DM-ADSC and N-ADSC transplants, reaching their lowest levels at ZT18. These results revealed that daily fluctuations in the mitochondrial genes differed in the phase between the N-ADSC and T2DM-ADSC transplants (Fig. 3c, d).
Daily fluctuations in mitochondrial function differed in phase and amplitude between the N-ADSC and T2DM-ADSC transplants
We explored the characteristics through which T2DM affected the daily fluctuations in mitochondrial function. Electron microscopic images of the mitochondrial structure showed that the crista structure was regularly aligned in the N-ADSC group. In contrast, the crista structure was destroyed in the T2DM-ADSC group, with fewer cristae being present (Fig. 4a). Therefore, we decided to analyze the daily fluctuations in the number of mtDNA copies. The N-ADSC group exhibited daily fluctuations in the number of mtDNA copies, reaching the lowest number at ZT10. In contrast, daily fluctuations were not observed in the T2DM-ADSC transplant (Fig. 4b). Difference in the mitochondrial membrane potential is critical for maintaining the production of mitochondrial ATP by the respiratory chain. Previous reports suggested that the mitochondrial membrane potential disappears after opening the mitochondrial permeability transition pore. This is followed by a flow of cytochrome C into the cytoplasm. In this study, a low molecular fluorescent dye JC-1 was used to observe the mitochondrial membrane potential. Differences in mitochondrial membrane potential prompt the accumulation of JC-1 in mitochondria. In addition, it alters the fluorescence properties from green fluorescence (wavelength: 530 nm) to red fluorescence (wavelength: 590 nm) due to the accumulation of the dye. Thereafter, the ratio of red fluorescence intensity to green fluorescence intensity was evaluated. The fluorescence intensity ratio of JC-1 mitochondrial accumulation, which reflects mitochondrial membrane potential difference, showed daily fluctuations in the N-ADSC transplant, peaking at ZT10. However, the T2DM-ADSC transplant did not exhibit daily fluctuations (Fig. 4c). Mitophagy is a system that selectively eliminates mitochondria that are degraded due to oxidative stress and DNA damage. In mitophagy, degraded mitochondria are isolated by autophagosomes and fused with lysosomes, where they are finally digested. In this study, we used the fluorescence intensity of Mtphagy Dye (which visualizes mitophagy) to evaluate daily fluctuations in mitophagy. In the N-ADSC transplant, the fluorescence intensity of the mitophagy-visualizing dye showed daily fluctuations, peaking at ZT10. In contrast, no daily fluctuations were observed in the T2DM-ADSC transplant (Fig. 4d). Moreover, signals regulating the daily fluctuations in mitochondrial genes and functions were examined by pathway analysis based on the results of next-generation sequencing. NF-kB was identified as a factor that plays a central role in the extracted signaling pathway (Fig. 5a). When daily fluctuations were evaluated using the NF-kB reporter assay, the results showed daily fluctuations in NF-kB reporter activity in the N-ADSC transplant, peaking at ZT10. Nevertheless, no daily fluctuations were observed in the T2DM-ADSC transplant (Fig. 5b).
This study investigated the circadian rhythms of clock genes and mitochondrial genes after stem cell transplantation. The results revealed the daily fluctuations in clock genes after transplantation of N-ADSC and T2DM-ADSC. These fluctuations were similar in phase and amplitude between the two groups. The data demonstrated that the clock genes in stem cells were synchronized with those in living organisms. Although mitochondrial genes and functions showed daily fluctuations only in the N-ADSC group, those was lost in the T2DM-ADSC group, revealing marked differences in phase and amplitude between the two types of transplants.
In this study, MTATP8P1 and NDUFA7_2 genes were identified to exhibit variations between two time points (ZT6 and ZT18). MTATP8P1 is a factor involved in mitochondrial ATP synthesis-coupled proton transport27. It is involved in periodic paralysis associated with cardiac myopathy and neuropathy28,29. NDUFA7_2 is a gene encoding a subunit of NADH:ubiquinone oxidoreductase (complex I), which is present in the mitochondrial inner membrane30. Complex-I regulates electron migration in the respiratory chain mediated by NADH30. However, prior to the present study, the circadian rhythms of MTATP8P1 and NDUFA7_2, had not been reported. The circadian rhythms of mitochondrial functions and NF-kB signaling revealed in this study may be useful as new markers for the efficiency of stem cell transplantation.
The validity and limitations of the methods utilized in this study are discussed below. Firstly, some clock genes exhibited rhythms, whereas others did not. The results of next-generation sequencing showed that the N-ADSC group had daily fluctuations in the clock genes PER1, PER2, PER3, CRY1, CRY2, and ARNTL (BMAL1), whereas daily fluctuations were not noted in CLOCK1, CLOCK2, CLOCK3, and CLOCK4 (Fig. 1b). Similarly, in the T2DM-ADSC group, PER1, PER2, PER3, CRY1, CRY2, and ARNTL (BMAL1) showed daily fluctuations, whereas daily fluctuations were not noted for CLOCK1, CLOCK2, CLOCK3, and CLOCK4 (Fig. 1c). In addition to the limit in the sensitivity of next-generation sequencing, the transcriptional regulatory sequence upstream of the clock genes may have been responsible for these differences. In mammals, basic helix-loop-helix protein (bHLH) transcription factors CLOCK and BMAL1 form a heterodimer, which binds to an E-box in the promoter regions of negative factor genes, such as PER and CRY31,32. This binding positively regulates the expression of genes, including PER and CRY33. Subsequently, the PER/CRY complex deactivates the transcriptional activity by CLOCK/BMAL1, thereby suppressing its own transcription33. This repetition forms an approximately 24-h cycle. In addition to this feedback loop, the responsive sequence of the clock genes and the secondary loop mediated by the Rev-responsive element and a D-box are functioning32,33. Given its limited detection sensitivity, it is impracticable to evaluate the circadian rhythms of the clock genes solely through next-generation sequencing. In this study, we performed an evaluation using quantitative PCR. The results revealed that the circadian rhythms of the clock genes PER2, CLOCK, CRY1, and BMAL1 were similar between the N-ADSC and T2DM-ADSC groups.
We also analyzed mitochondrial genes and functions in primary culture cells derived from transplants. The circadian rhythms of the clock genes are regulated at an approximately 24-h cycle by the central clock genes in the SCN33. Regulation by the central clock is abolished following tissue extraction and cell proliferation by primary culture. Accordingly, the clock genes fail to produce synchronized rhythms unless shocks are induced with high-concentration serum or dexamethasone34. In this study, we evaluated the impact of extraction time without inducing such shocks. Therefore, changes were likely smaller than those observed after shocks induced by high-concentration serum or dexamethasone. However, an advantage of this evaluation system is the ability to detect differences based purely on the extraction time.
In this study, human adipose-derived ADSCs were transplanted into immunodeficient mice, the results of this study may not correlate with those reported after stem cell transplantation into humans. In particular, signaling from the SCN35, the central core of the clock genes, has been demonstrated in mice in this model. Therefore, the regulation by the central clock after stem cell transplantation may differ between mice and humans. This is a limitation of this study. Currently, it is difficult to monitor clock genes in transplanted tissue in humans based on the available technology. Hence, the present findings may be beneficial in predicting circadian rhythms in humans post-transplantation.
The present findings may contribute to clarifying the daily fluctuations in clock genes and mitochondrial genes following stem cell transplantation. This study revealed that the circadian variations in mitochondrial genes and functions were independent of those observed in clock genes. Moreover, T2DM-ADSC exhibited circadian variations in mitochondrial genes and functions that were different from those of N-ADSC. These findings suggest that diabetes mellitus may be related to the circadian variations of mitochondria in stem cells. In future studies, we intend to clarify the molecular regulatory mechanism underlying the circadian variations in mitochondrial genes and functions in T2DM-ADSC. In particular, we hope to uncover the regulatory mechanism of NF-kB signaling, which was investigated in this study. Finally, we also wish to elucidate the circadian rhythms of mitochondria following stem cell transplantation into humans.
Materials
The materials and instruments used in this study are listed in Supplementary Table 1.
ADSC transplantation model
The ADSC transplantation model used in this study was established in our preceding studies18,19. Briefly, it was created by subjecting ADSCs of mice lacking T-cell function (BALB/cAJCL-nu/nu) to spheroid culture using Matrigel medium and subcutaneously transplanting the 1 × 107 cells into the dorsum of mice. Five-week-old female BALB/cAJCL-nu/nu mice were purchased from CLEA Japan Incorporated (Tokyo, Japan) and raised under normal breeding conditions with a 12-h light/ a 12-h dark cycle. To analyze the daily fluctuations in clock genes, the lighting start time (start point of the light period) was regarded as ZT0 (signifying 0 h in Zeitgeber time). At 4-h intervals from ZT2, stem cell transplants were extracted.
N-ADSCs, originally established by primary culture of adipose tissue extracted from a 23-year-old healthy female without any underlying condition, were purchased from LONZA KK (lot number 18TL212639; city, country). T2DM-ADSCs, originally established by primary culture of adipose tissue extracted from a 39-year-old female with T2DM patient, were also purchased from LONZA KK (lot number 1F4619). Samples were collected 1 week later transplantation and used in the ensuing experiments.
The study was conducted according to the guidelines for the Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions and approved by the Institutional Review Board of Sanyo-Onoda City University (protocol code 2020-8-A, and date of approval 12 March 2020). Also, the study was conducted according to the ARRIVE guidelines.
Primary ADSCs were purchased from Lonza (Adipose-Derived Stem Cells (Lonza, SC, USA)). This study used ADSCs that were ethically approved by Lonza. The ADSCs consent form was obtained at Lonza, where the cells were purchased.
Next-generation sequencing
Total RNA was extracted from the collected transplants using a RNeasy Mini Kit. Poly (A) RNA was extracted from 100 ng of total RNA and fragmented using the NEBNext Poly(A) mRNA Magnetic Isolation Module and the NEBNext Ultra II RNA Library Prep Kit for Illumina. The fragmented poly (A) RNA was subjected to reverse transcription for the preparation of cDNA using the NEBNext First Strand Synthesis Enzyme Mix of the NEBNext Ultra II RNA Library Prep Kit for Illumina. Subsequently, the NEBNext Adaptor was added. The prepared cDNA was amplified by polymerase chain reaction (PCR) to create a library. The cDNA domain (75 bp) and barcode sequence were analyzed by fragment analysis using the Illumina NextSeq.
Quantitative PCR
The collected transplants were homogenized, and mRNA was extracted using a RNeasy Mini Kit. Reverse transcription was performed using a PrimeScript® RT reagent Kit to prepare template cDNA. Primers were purchased from the TAKARA Perfect Real Time Primer Support System. The primers used are listed in Supplementary Table 2. The template cDNA, PCR enzyme (SYBR Premix Ex Taq™ II), and forward and reverse primers were mixed for the subsequent reaction. The reaction was performed using a quantitative PCR device (StepOne-Plus-01). In real-time PCR, the conditions were: 95ºC for 30 s (Step 1), followed by 95ºC for 5 s and 60ºC for 60 s (Step 2) for 40 cycles. The expression levels of target genes were calculated using calibration curves, and the relative expression was calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard.
Observation of mitochondria using electron microscopy
Cells were fixed with 2% glutaraldehyde and 2% osmium solution, dehydrated with ethanol, and embedded in epoxy resin. Sections (thickness: 80–90 nm) were prepared using an ultramicrotome and placed on 200 meshes. Subsequently, the sections were stained with 2% uranium acetate and lead stain solution. Finally, the mitochondria were observed using a scanning microscope (H-7600 TEM; HITACHI) at 100 kV.
Mitochondrial DNA (mtDNA) copies
The number of mtDNA copies was determined by four different genes (NADH dehydrogenase subunit 1 [ND1], solute carrier organic anion transporter family member 2B1 [SLCO2B1], ND5, and serpin family A member 1 [SERPINA1]) using the Human mtDNA Monitoring Primer Set. Genomic DNA was extracted by homogenizing transplants and using a NucleoSpin Tissue kit. The genomic DNA, four different primers in the Human mtDNA Monitoring Primer Set, and a PCR enzyme (SYBR Premix Ex Taq™ II) were mixed for subsequent reaction. The reaction was performed using a quantitative PCR device (StepOne-Plus-01) under the following conditions: 95ºC for 30 s (Step 1), followed by 40 cycles of 95ºC for 5 s and 60ºC for 30 s (Step 2). The number of mtDNA copies was calculated as the mean of 2ΔCt from the difference in the cycle threshold values of ND1/SLCO2B1 and ND5/SERPINA1.
Analysis of the mitochondrial membrane potential
The mitochondrial membrane potential was analyzed using a JC-1 MitoMP Detection Kit. After treating transplants with trypsin and STEMxyme 2-collagenase, dissociated cells were primary cultured and inoculated into an eight-well culture slide and incubated overnight under normal culture conditions in an incubator adjusted to 37ºC and 5% CO2. Following the addition of 100 µl of JC-1 working solution (4 µM), the sample was incubated for 30 min at 37ºC and 5% CO2. After washing the sample by HBSS, an imaging buffer solution was added. Finally, the sample was observed with a fluorescent microscope (green and red fluorescence at 488 nm and 561 nm wavelength, respectively).
Mitophagy analysis
Mitophagy was analyzed using the Mtphagy Dye and Lyso Dye of the Mitophagy Detection Kit. After treating transplants with trypsin and STEMxyme 2-collagenase, primary culture cells were prepared. Cells were inoculated into a 96-well plate and incubated overnight under normal culture conditions in an incubator adjusted to 37ºC and 5% CO2. After washing the sample twice with a serum-free medium, the Mtphagy Dye working solution was added at 100 nM and incubated for 30 min at 37ºC and 5% CO2. To observe the colocalization of Mtphagy Dye and lysosome, 1 µM Lyso Dye working solution was added, and the sample was further incubated at 37ºC and 5% CO2. After washing the sample by Hanks` HEPES buffer, staining of the mitophagy dye (Mtphagy Dye) and lysosome dye (Lyso Dye) were observed with a fluorescent microscope (green and red fluorescence at 488 nm and 561 nm wavelength, respectively). Following the confirmation of colocalization, the red fluorescence from the mitophagy dye (Mtphagy dye) was measured at 561 nm wavelength using a fluorescence plate reader.
Nuclear factor-kB (NF-kB) reporter assay
An NF-kB Secreted Alkaline Phosphatase Assay Kit was used to evaluate the NF-kB activity. Transplants were treated with trypsin and STEMxyme 2-collagenase to prepare primary culture cells. Cells were inoculated into a 96-well plate and incubated overnight under normal culture conditions in an incubator adjusted to 37ºC and 5% CO2. Cells were inoculated into a six-well culture plate at 5 × 105 cells/well and incubated until they reached 80% confluence. A mixture of the pNF-kB/SEAP plasmid DNA (4 µg) and Lipofectamine LTX (10 µl) was added at 24-h intervals, and the cells were incubated for 72 h. Cells were collected and analyzed at 405 nm wavelength using the Secreted Alkaline Phosphatase (SEAP) assay to quantify the NF-kB reporter activity based on calibration curves.
Statistical analysis
Results are presented as the mean ± standard deviation. Comparison between multiple groups was performed using two-way ANOVA with Tukey–Kramer post hoc tests or Holm post hoc tests, with P-values < 0.05, denoting statistical significance.
Conflict of Interest Statement (COI)
The authors indicate no potential conflicts of interest on this paper.
Ethics approval and consent to participate
The study was conducted according to the guidelines and the regulations for the Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions and approved by the Institutional Review Board of Sanyo-Onoda City University (protocol code 2020-8-A, and date of approval 12 March 2020). Also, the study was conducted according to the ARRIVE guidelines.
Primary ADSCs were purchased from Lonza (Adipose-Derived Stem Cells (Lonza, SC, USA)). This study used ADSCs that were ethically approved by Lonza. The ADSCs consent form was obtained at Lonza, where the cells were purchased.
Funding
This work was supported by The Uehara Memorial Foundation to M.H.
This work was supported by The Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for KAITAKU (20K20590) to M.H.
Author Contribution Statement
Conceptualization (M.H.), Data Curation (M.H.), Formal Analysis (M.H., Y.M., K.W.), Funding Acquisition (M.H.), Investigation (M.H.), Methodology (M.H., Y.M.), Project Administration (M.H.), Resources (M.H.), Software (M.H.), Supervision (M.H.), Validation (M.H.), Visualization (M.H., Y.M.), Writing – Original Draft Preparation (M.H.), Writing – Review & Editing (M.H., Y. T., K.U.)
Data availability
The datasets generated during the current study are available in the Harvard Dataverse repository, https://doi.org/10.7910/DVN/DWOLNR.
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Table 1 Top six categories of the fluctuating genes in adipose-derived mesenchymal stem cells derived from a healthy individual and a patient with type 2 diabetes mellitus transplants determined by next-generation sequencing
No competing interests reported.