Isolation and culture associated with NHSCs from human UCB
UCB was purchased from South Texas Blood & Tissue Center (San Antonio, TX, USA). MNCs had been isolated from UCB using Ficoll-Paque Premium density mass media (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) as defined previously [ 14 ]. MNCs were suspended in “ growth media” containing alpha-minimal essential media (α -MEM; Life Technologies, Grand Island, NY, USA), penicillin (100 U/ml), streptomycin (100 μ g/ml; Biofluids, Rockville, MARYLAND, USA), 15% pre-selected fetal bovine serum (FBS; Becton Dickinson, Franklin Lakes, NJ, USA), l -glutamine (4 mM), and fundamental fibroblast growth factor (bFGF; 4 ng/ml) and seeded at 10 6 MNCs/cm 2 onto TCP that was either uncoated or coated with fibronectin [ 22 ] or even coated with ECM made by human BM stromal cellular material [ 19 , 20 ]. After seeding, MNC cultures were incubated for 72 h at 37 ° C making possible attachment of the cells and then washed twice with phosphate-buffered saline (PBS) to remove non-adherent cells. After washing, development media were added and half media changes had been performed every 3 days. The adherent cells had been cultured to 70– 90% confluence in a humidified environment at 37 ° C. Adherent cells (passage (P)1) were detached from TCP surfaces using trypsin or maybe the ECM surface using collagenase. An aliquot of the tissues (P1) was used to determine the phenotype of the inhabitants using flow cytometry (see below), while the remaining tissue were either used immediately or stored in liquid nitrogen. Over 100 UCB samples were obtained from the cells bank and processed for this study. A subset associated with 50 of these samples was used in the current study simply because they had been more completely analyzed for cell phenotype.
Human BM cells
Newly isolated human BM-MNCs (containing MSCs), obtained from 20- in order to 30-year-old donors and purchased from ALLCELLS (Emeryville, CALIFORNIA, USA), were used to prepare BM stromal cell-derived ECM (BM-ECM) as described previously [ 20 ]. Previously, all of us demonstrated that BM-ECM is superior to uncoated TCP being a culture surface for human BM-MSCs and promotes connection, expansion, and “ stemness” of these cells [ 20 ]. For the present study, human BM-MNCs or MSCs had been cultured on this ECM for comparison with UCB-derived tissue.
Anti-CD34, CD45, CD105, and CD146 antibodies were purchased from R& D Systems (Minneapolis, MN, USA). Anti-CD29 and SSEA-4 antibodies were purchased from BD Bioscience (San Jose, CA, USA). Single-cell suspensions (1– 3 × 10 5 ) were incubated meant for 30 min at 4 ° C in 100 μ l of specific antibody (10 μ g/ml). Antibody-labeled cells were washed twice with staining barrier (PBS containing 5% fetal calf serum (FCS) plus 0. 01% sodium azide) and incubated in 20 μ g/ml of FITC-conjugated goat anti-mouse IgG pertaining to 20 min at 4 ° C. Cells had been then washed two times with staining buffer and possibly analyzed immediately or fixed with 1% paraformaldehyde plus analyzed within 96 h using a Becton Dickinson FACStar plus (Franklin Lakes, NJ, USA) flow cytometer with 10, 000 events collected for each sample. The percentage of positive cells was dependant on fluorescence-activated cell sorting (FACS). All assays were operate in parallel using cells stained with isotype IgG as a negative control.
Determination of cell human population doubling time (PDT)
UCB-derived cells (P3– 5), previously maintained on ECM, had been seeded (4 × 10 3 cells/cm 2 ) onto ECM-coated plates in growth media and cultured for up to 9 days. At harvest, adherent cells were detached along with collagenase and the number of live cells determined in triplicate. The average number of cells for each time point was plotted against time to generate a growth curve. PDT was identified as described previously [ 23 ].
UCB-derived cells (P2), previously maintained upon ECM, were seeded (2. 5– 7. 5 × 10 4 cells/cm 2 ) onto chamber slides and classy for 2 days in growth media. The cells had been fixed with 4% formaldehyde for 30 min with room temperature, washed with PBS, and then blocked intended for 1 h using 5% normal goat serum that contains 0. 1% bovine serum albumin (BSA). The cells had been stained by incubation with primary antibodies (1: hundred dilution) against OCT4, TRA-1-60, and SSEA-4 for 30 min at 4 ° C. Non-specific isotype bunny IgG (1: 100 dilution), mouse IgM (1: one hundred dilution), and mouse IgG (1: 100 dilution) had been used as negative controls, respectively. After washing along with PBS, samples were incubated for 20 min with all the appropriate FITC-conjugated secondary antibody. Specimens were mounted within DAPI-containing medium (Vector Laboratories, Burlingame, CA, USA) as well as the cells visualized using an Olympus FV500 Fluoview confocal microscope equipped with image analysis software to quantify fluorescence strength (Olympus America, Inc., Melville, NY, USA).
Determination associated with colony-forming unit fibroblast and osteoblast numbers
To determine the number of colony-forming unit fibroblast (CFU-F) colonies in the initial MNC population, freshly isolated UCB cells were seeded at low seeding density (1 × 10 5 MNCs/cm 2 ) onto ECM and uncoated or fibronectin-coated TCP surfaces and cultured at 37 ° C in growth media. Half media adjustments were performed every 3 days. After 1 30 days, the cells were fixed and stained with crystal purple to determine CFU-F number. To assess CFU osteoblast (CFU-OB) colony formation, CFU-F colonies were maintained for an extra 25 days in osteoblast differentiation medium (growth mass media supplemented with 100 nM dexamethasone (Sigma), 50 μ M l -ascorbate-2-phosphate (Wako Chemicals, Richmond, VA, USA), and 10 mM glycerol 2-phosphate). CFU-OB colonies were counted after staining along with von Kossa.
Adipogenesis and myogenesis
UCB-derived cells (P1– 3), previously managed on ECM, were seeded (4 × ten 3 cells/cm 2 ) onto ECM-coated plates in growth media plus cultured to confluence. To study adipogenesis, the media had been changed at confluence to differentiation media (DMEM that contains 10% FBS, 0. 5 mM IBMX, 1 μ M dexamethasone, 10 μ g/ml insulin, 100 μ M indomethacin) [ 24 ] and one-half mass media changes were performed every 5 days. After 20 days in culture, adipocytes were visualized by discoloration with Oil Red O. To study myogenesis, the press were changed at confluence to differentiation media (DMEM containing 10% FBS, 5% horse serum, 10 -7 M dexamethasone, 50 μ Mirielle hydrocortisone) [ 24 ] and one-half media adjustments were performed every 5 days. After 25 times in culture, myotubes were identified after staining along with hematoxylin and eosin (H& E).
Cardiomyogenesis plus angiogenesis
Cardiomyogenesis was caused as described previously [ 25 ]. Briefly, UCB-derived tissue (P2) or BM-MSCs (P2), previously maintained on ECM, were seeded (10 6 cells/cm 2 ) onto TCP and classy for 2 days in standard growth media, then an additional 2 days in media containing 5 μ M 5-azacytidine (Sigma). Subsequently, the cells were switched in order to differentiation media (DMEM containing 10% FBS, 10 – 4 M ascorbic acid (Sigma) and 10 ng/ml transforming growth factor (TGF)-β ) and cultured for up to 25 days with media changed every 4 days. The expression of cardiac troponin T type 2 (TNNT2) was quantified using TaqMan polymerase chain reaction (PCR) after culture for 25 days in either growth or differentiation media.
To study angiogenesis, UCB-derived cells (P2), previously maintained on ECM, were seeded (3 × 10 3 cells/cm 2 ) onto six-well TCP plates covered with Matrigel™ (BD Bioscience, San Jose, CA, USA) in 1 ml endothelial differentiation media (EGM-2; Lonza, Switzerland) and cultured for 7 days at 37 ° C. Media changes were performed every 3 days. Capillary formation was documented with brightfield photomicrographs. Baboon endothelial progenitor cells (positive controls) were i implore you to provided by Dr . Qiang Shi (TBRI, San Antonio). BM-MSCs (P2) were used as negative controls.
Solitude of RNA and gene expression analyses using current PCR
Total RNA had been extracted from cultured cells using Ultraspec™ RNA (Biotecx, Houston, TX, USA) [ 19 ]. RNA (2 μ g) was reverse-transcribed using a High Capacity cDNA Store Kit (Applied Biosystems, Foster City, CA, USA). The particular transcripts of interest, including housekeeping genes (GAPDH or β -actin), were amplified from the cDNA by real-time PCR using TaqMan Universal PCR Master Mix and TaqMan based primer and probe sets (TaqMan gene Manifestation assays, Applied Biosystems). Amplification and detection were performed with an ABI Prism 7500 Sequence Detection System (Applied Biosystems) and gene expression quantified by subtracting the particular GAPDH threshold cycle (Ct) value from the Ct associated with the gene of interest and expressed as 2 – Δ Ct . RNA isolated through hES (ESI6) was kindly provided by Dr . Peter M. Hornsby (UTHSCSA) and served as a positive control.
In vivo implant assay
Cells (1 × 10 6 ) were loaded onto Gelfoam (Pharmacia & Upjohn Company, MI, USA) or hydroxyapatite/tricalcium phosphate (HA/TCP) contaminants (Zimmer Inc, Warsaw, IN, USA) and implanted subcutaneously into the dorsal surface of 10-week-old immunodeficient beige rodents (NIH-bg-nu-xid; Harlan Sprague Dawley, Indianapolis, IN, USA) [ 19 ] according to an approved IACUC protocol on UTHSCSA. After 8 weeks, implants were harvested, set, decalcified (HA/TCP implants only), and embedded in paraffin. Sequential sections were processed and stained with H& E. Bielschowsky’ s silver stain was used to determine nerve [ 26 ]. To determine the origin of neotissues produced during implantation, a section adjacent to the H& E discolored section was stained with an antibody specific for individual nuclear ribonucleoprotein (Millipore, Billerica, MA, USA) [ 27 ].
Green fluorescent protein labeling
To prepare ECM-adherent UCB cells stably expressing green neon protein (GFP), commercially available Lentiviral particles (GeneCopoeia Lentifect™, LP-EGFP-LV105-0200) were used as described in the manufacturer’ s i9000 instructions. Cells, labeled with an efficiency of > 70%, were sorted using flow cytometry and GFP(+) tissues (95% positive) were expanded for the experiments.
Muscle reconstruction using a cryoinjury animal model
A model of muscle cryoinjury was established in homozygous immunodeficient RAG2 – /– , γ c– /– mice (Taconic, Germantown, NY, USA) that were preserved as a breeding colony and housed in a temperature-controlled, air-filtered room with a 12-h light-dark cycle. All procedures had been performed according to an approved IACUC protocol at UTHSCSA.
Adult mice (8 in order to 12 weeks old) were anesthetized and the gluteus maximus muscle exposed bilaterally. The oval shaped end (4 × 6 mm) of a 12 inch lengthy copper probe was immersed in liquid nitrogen and applied to the muscle for 20 s. Following cryoinjury, 10 6 UCB-NHSCs were hanging in 35 μ l 50% Matrigel™ (1: one Matrigel™ to DMEM) and 10 μ l aliquots of this suspension were implanted intramuscularly in three areas within the injured area, and then the skin was closed. Automobile (35 μ l 50% Matrigel™ ) and epidermis fibroblasts (10 6 cells within 35 μ l 50% Matrigel™ ) were utilized as controls. After 28 days, the animals had been euthanized and muscle tissue harvested for histology or take frozen in liquid nitrogen and stored at – 80 ° C.
To successfully predict the isolation of NHSCs from UCB tissue, we initially employed a general linear regression model using MATLAB Statistics and Device Learning Toolbox. The independent variables considered were: UCB sample volume, time since delivery in hours (time between tissue harvest and cell isolation in the laboratory), and cell surface phenotypic markers. The model pointed out that all of the variables, except CD146, failed to identify a mixture capable of predicting success. Next, we used a stepwise approach which excluded variables that failed to enhance our own ability to predict the successful isolation of UCB cellular material. We began with the generalized linear regression model and after that, in stepwise fashion, tested whether removing/adding variable(s) enhanced predictive power. The final model was arrived at by getting to a point where no single backward or forward step enhanced the model’ s predictive accuracy.
All data are presented as the mean ± standard deviation ( and = 3 or 6 depending on the experiment). All results were reproduced in at least six independent tests with cells from six or more donors. For within vivo studies, a total of 18 implants, using cellular material from six or more donors, were performed. Statistical studies were performed with the Student’ s t test or one-way evaluation of variance (ANOVA) with significance set at p < 0. 05.