MSC collection and characterization
Umbilical cord segments immersed in phosphate-buffered saline (Lonza, MD) were minced into 1–2 mm3 fragments and aligned at regular intervals in 10 cm culture dishes until they were firmly attached to the bottom of the dish for at least 30 minutes and expanded in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal bovine serum at 37°C and 5% O2 and 5% CO2. Culture media was replaced 2–3 days after initial plating and then every 2 days. Adherent cells were characterized as MSCs according to the International Society of Cell Therapy guidelines by using immunocytochemistry to confirm expression of stem cell markers including CD105 and CD90 (Fig. 7A). Mesenchymal differentiation ability of these cells was tested by inducing cells to differentiate in media supplemented with 100 µM indomethacin, 0.5 mM IBMX, and 10 µg/mL insulin and staining with Oil Red O (Millipore Sigma, VT) to confirm their differentiation into adipocytes. Once the fibroblast-like adherent cells reached 80–90% confluence they were removed from the culture dishes using 10% trypsin and passaged further or cryopreserved for future use.
OCR assay
Preliminary studies of MSC bioenergetics were used to determine that a seeding density of 30,000 MSCs/well produced optimal (100 pmol/min) basal OCR rates best suited for performing mitochondrial stress tests (Fig. 7B). Therefore, 30,000 MSCs were seeded per well into Seahorse™ XF96 Cell Culture Microplates (Agilent) and allowed to adhere for up to 24 hours on the day before the assay. On the day of the assay, MSCs were washed once and incubated in DMEM supplemented with 10 mM glucose and 2 mM L-glutamine. After calibration of the Seahorse XF96 Analyzer, the Seahorse™ XF96 Cell Culture Microplate was inserted and basal oxygen consumption rates (OCR) were determined followed by further OCR measurements after the addition of mitochondrial inhibitors Oligomycin (1 µM), carbonyl cyanide-p-triflouromethoxyphenylhydrazone (FCCP, 1 µM), rotenone/antimycin ( 0.5 µM/1 µM) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD)/ascorbic acid (0.5 mM/2 mM) as described previously.(8, 57)
ECAR assay
ECAR, measured using a Seahorse™ XF96 analyzer, was used as a surrogate for anaerobic glycolysis. 30,000 MSCs were seeded per well in Seahorse™ XF96 Cell Culture Microplates (Agilent) and allowed to adhere for up to 24 hours on the day before the assay. On the day of the assay, cells were washed with XF base medium and incubated for 60 minutes at 37°C in Seahorse XF Base medium supplemented with 5 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate, and 0.5 mM HEPES buffer, pH 7.4 in the absence of CO2. Basal ECAR was first measured followed by addition of glucose (10 mM) to measure glycoysis-associated ECAR, oligomycin (2 µM) to inhibit mitochondrial oxidative phosphorylation to measure ECAR associated with maximal glyolysis (glycolytic capacity) and 2-deoxyglucose (100mM) to confirm suppression of ECAR measurements due to inhibition of glycolysis, as described previously.(58)
PER assay
As bulk acidification of extracellular medium measured by ECAR are not specific to glycolysis but also includes acidification caused by CO2 released through mitochondrial phosphorylation, glycolytic rate assays were also performed to measure glycolysis-specific PER. 30,000 MSCs were seeded per well in Seahorse™ XF96 Cell Culture Microplates (Agilent) and incubated in Seahorse XF Base medium supplemented with 5 mM glucose, 2 mM glutamine, 1 mM sodium pyruvate. Followed by basal measurements of PER, MSCs were next treated with simultaneous addition of antimycin A and rotenone (0.5 µM/1 µM) to inhibit mitochondrial oxidative phosphorylation and subsequent production of carbonic acid produced from the CO2 generated by the TCA cycle. PER measurements during this phase enable determination of the mitoPER (PER from mitochondrial respiration) and compensatory glycoPER (PER from glycolysis). Finally, 2-deoxyglucose (100mM) was injected to inhibit glycolysis and provide qualitative confirmation that proton efflux measured in the experiment was secondary to glycolysis.(59)
ATP assay
A luminescent ATP Detection Assay Kit (ab113849, Abcam, MA) was used to measure MSC ATP. Briefly, luciferase enzyme and luciferin were added to MSC lysates, and the luminescence emitted was measured using a SpectraMax™ i3 reader (Molecular Devices, CA) and quantified using standard curves for ATP generated by following manufacturer’s instructions.
Quantitative PCR
RNA from cultured cells exposed to normoxia (5%) or hyperoxia (85%) for 24 hours was isolated with RNeasy Plus Mini Kit (Qiagen, Cat. No.74136).The concentration and purity of the RNA were measured checked using SpectraMax i3x (Molecular Devices). Then a total of 1µg of RNA was reverse-transcribed to cDNA using iScript Reverse Transcription Superrmix (Bio-Rad, Cat. No.1708840), following the manufacturer’s instructions and the cDNA was used as a template in the subsequent qPCR which was performed using TaqMan Fast Advanced master Mix (Thermo Fisher Scientific. Cat. No. 4444556) with TaqMan gene expression assays for PINK1 (Hs00260868_m1) according to the manufacturer’s instruction of Taqman Fast Advanced Master Mix on the Bio-Rad CFX96 system.(60) All qPCR were carried out using five independent samples in duplicate. To correct the differences in the amount of cDNA loading into qPCR reaction wells, Eukaryotic 18S rRNA Endogenous Control (Thermo Fisher Scientific. Cat. No. 4310893E) was used to normalize the expression levels of the target genes. qPCR data was analyzed by applying the comparative Ct method (ΔΔCT). The results were presented as the fold change in mRNA expression for targeted genes relative to controls.
Western blotting
MSCs exposed to normoxia (5%) or hyperoxia (85%) for 24 hours were lysed in RIPA buffer (Alfa Aesar. Cat. No. J62885) and the lysate was centrifuged at 6,000g for 10min at 4ºC, then the supernatant was mixed with 4x Laemmli sample buffer for western blotting according to standard procedures. 30 to 60 µg of total protein from the samples were separated on 4–20% Criterion TGX Gels (Bio-Rad, Cat. No. 5678093), transferred to PVDF membranes (Trans-Blot Turbo System, Bio-Rad) and probed with antibodies against PINK1 (ab23707), Aconitase (ab181153),TOM20 (ab56783, Abcam, MA) and B-Actin (Cell Signaling Technology, 5125S) as loading control.(61) Membranes were developed using Clarity ECL Substrate (Bio-Rad) and visualized with ChemiDoc imaging System. Densitometry analysis of band intensities was performed using ImageJ software.
TEM imaging
MSCs exposed to normoxia (5%) or hyperoxia (85%) for 24 hours were detached from plates using 0.25% Trypsin and spun down at 1,000g for 5min to remove media. The cell pellet was fixed in 0.1M Sodium Cacodylate Buffer pH 7.4 with 1% Osmium tetroxide (EMS) for 1h at RT, then dehydrated through a series of graded ethyl alcohols from 50 to 100%. After the infiltration process in propylene oxide, the specimens were embedded in a fresh 100% embedding media and polymerized at 60ºC overnight. For thin sections, the appropriate blocks are cut using a diamond knife (Diatome, Electron Microscopy Sciences, Hatfield, PA) at 70-90nm (silver to pale gold using color interference) and sections were then placed on copper grids. After drying, the sections were stained with the heavy metals uranyl acetate and lead citrate for contrast and then viewed on a Tecnai Spirit 120kv TEM (FEI, Hillsboro, OR). Digital images were taken with an AMT BioSprint 29.(62)
IHC of umbilical cord sections
IHC was performed on 10% formalin – fixed and paraffin - embedded (FFPE) 5 µm sections of umbilical cords. Tissue sections on the slides were deparaffinized first with xylene and then rehydrated with alcohol in a graded fashion. Endogenous peroxidases were blocked with 3% hydrogen peroxide and antigen was retrieved by placing the slides in a thermostatic bath of sodium citrate buffer solution for 15–30 minutes at 98°C and then allowing them to cool down to room temperature. The tissue sections were then treated with 1% BSA for an hour to block non- specific endogenous antibodies and next with anti CD-105 rabbit monoclonal antibody (Abcam, CA) at 1: 200 dilution after which they were left overnight at 4°C. After rinsing the antibody incubated tissue sections, the antigen antibody complexes in tissue sections were visualized with diaminobenzidine (DAKO and DAB substrate kit) reaction that resulted in brown staining of the membrane. Images of stained tissue sections were captured using a Nikon-T1E microscope (Nikon, NY). 20 images of umbilical cord sections per infant were analyzed to measure the area of each image occupied by cells positive for stem cell surface markers using ImageJ software.
Proliferation assay
A CyQUANT™ MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Assay Kit (Thermofisher, MA) was used to measure MSC proliferation. The assay is based on the principle that redox potential of viable mammalian cells causes the conversion of water-soluble MTT to formazan that can be analyzed colorimetrically. 1x105 MSCs/well were seeded in 96-well plates and incubated with 100 µL of culture media and 10 µL of 12 mM MTT solution for 4 hours. 100 µL of sodium dodecyl sulfate was next added to each well to solubilize the formazan. Each microplate was incubated for a further 12 hours at 37°C and absorbance was measured at 540 nm using a SpectraMax™ reader. 10 µL of the MTT stock solution to 100 µL of medium alone was included as a negative control for each sample.(63)
Apoptosis assay
Caspase-3 activity in MSC lysates was determined using the Caspase-3 Colorimetric Assay Kit (BioVision, CA).Following manufacturer’s instructions, 5x106 MSCs were lysed and centrifuged at 10000×g for 1 min and the supernatant, reaction buffer (10 mM DTT) and Asp-Glu-Val-Asp–p-nitroanilide (a substrate for caspase-3) were incubated for 2 h at 37°C and transferred to 96-well plates. Absorbance at 405 nm was read by a SpectraMax™ reader.(64)
Aconitase activity
To measure mitochondrial aconitase activity, an aconitase activity kit (Abcam, CA) was used as per manufacturer’s instructions. Briefly, MSCs in six-well plates were exposed to normoxia or hyperoxia for 24 hours. After treatment, cells were homogenized in 100 µL of assay buffer provided and spun at 20,000 g for 15 min at 4°C to separate mitochondrial fractions. 300 µg of isolated mitochondria per sample were re‐suspended with 100 µL of assay buffer and then incubated for 1 h with the isocitrate substrate mixture at 25°C and absorbance at 240 nm was recorded for 30 min using a SpectraMax™ reader. Catalytic activity of aconitase was determined by measuring the rate of formation of cis-aconitate as detected by increase in the absorbance.(65)
Statistical analysis
Student’s t-test (parametric), Wilcoxon rank sum test (non-parametric) and Chi-Square test or Fisher’s exact test (categorical) were used to test for differences between individual pairwise comparisons. Correlation between continuous variables was evaluated using Pearson’s test. All analyses were performed using R 4.0.2.(66) Results are expressed as mean ± SD or median (IQR). P < 0.05 was considered significant.To detect a difference of 30 ± 15 pmol/min/30k cells (mean ± SD) difference in MSC basal OCR (based on OCR measurements in umbilical venous endothelial cells obtained from ELBW infants in our previous study) at 5% level of significance (α = 0.05), and 80% power, we estimated that we would need a sample size of 15 infants per group.(8)
Study Approvals
All protocols were approved by the Institutional Review Board of the University of Alabama at Birmingham (UAB). Infants were enrolled after appropriate informed consent was obtained from their mothers before or within 6 hours after birth. All procedures and experiments conducted in the study were carried out in accordance with all relevant institutional guidelines and regulations.