Reduction of Mitochondrial Superoxide By Carbon Nanotube Scaffolds Enhances Ex Vivo Expansion of Human Cord Blood Progenitor Cells

Hematopoietic stem cell transplantation (HSCT) with umbilical cord blood (UCB) is primarily for pediatric patients, as UCB has insufficient cell dosage for adult HSCT. The use of biomimetic scaffolds amongst other methods has been investigated for ex vivo expansion of UCB hematopoietic stem & progenitor cells (HSPC) prior to transplantation. One such scaffold that we identified is surface functionalized single walled carbon nanotubes (f-SWCNT). In this study, we report how HSPC survival, molecular changes & ex vivo expansion are dependent on various surface functional group of the f-SWCNT. Furthermore cotransplantation of f-SWCNT expanded & non-expanded cells from the same UCB enhanced human cell engraftment in immunodeficient xenograft models.

UCB-mononucleated cells (MNC) cultured in serum free medium with basal cytokines were exposed to dispersed f-SWCNT with varying surface functional groups namely carboxylic acid (–COOH), amide (–O-NH₂) & polyethylene-glycol (–PEG) at concentrations of 0 to 1.0 mg/ml. UCB-MNC cultures with cytokines served as control. The effects of f-SWCNT treatment were measured based on cell viability; phenotypic-based HSPC number; & functional in vitro & in vivo assays.

Short term cultures showed that CD45⁺ viability rose linearly with increasing concentrations for each type of the f-SWCNT (R²>0.770). Peak viability was observed at a concentration of 1.0 mg/ml with –COOH (94.0±0.3 %) giving the maximal support compared to –PEG (85.3±3.4 %), –O-NH₂ (78.3±0.8 %) & control (73.5±1.9 %) (n=6; p<0.01). Caspase activation analysis of CD45⁺ cells showed that –COOH significantly reduced active caspases 3/7, 8 & 9 followed by –PEG, –O-NH₂ & control (p<0.01; n=3). Exposure of the CD45⁺ cells to the f-SWCNT decreased the level of mitochondrial superoxides with optimal effect in –COOH (0.7±0.1 %) followed by –PEG (24.6±0.6 %), –O-NH₂ (40.5±1.8 %) & control (95.3±1.0 %) (p<0.01; n=3). A strong negative correlation (R = –0.86) existed between cell viability & mitochondrial superoxides.

In 11-days expansion system, –COOH gave an optimal expansion of CD45⁺CD34⁺CD38^–progenitors (492.9±66.7 folds) & CFU-GM (72.1±9.0 folds) compared to –PEG (476.0±100.3 & 38.8±6.4 folds), –O-NH₂ (429.1±65.3 & 49.5±4.6 folds) and control (156.9±9.3 & 26.9±1.4 folds) (p<0.05; n=3). Similar trends were observed for myeloid (CD34⁺CD13⁺CD33⁺) progenitor expansion (p<0.05; n=6). Antibody array of secreted proteomics in –COOH augmented cultures showed significant down regulation of chemokines (CC or CXC), inflammatory & differentiation promoting cytokines compared to control (p<0.01; n=3). Cytokines such as Eotaxin-3, IL-3, IL-10, IGF-1, SCF, IGFBP2, IGFBP3 & SDF-1 were detected at a significantly high level in the –COOH cultures compared to the control suggesting a favorable environment for the HSPC (p<0.001; n=3).

After 16 weeks of transplanting f-SWCNT-COOH expanded (0.02±0.02 %; n=6) or cytokine only expanded (0.02±0.02 %; n=6) UCB-MNC at an equivalent cell dosage of 2.5x10⁷ cells/kg to sub-lethally irradiated, immunodeficient NSG mice resulted in lower (p=0.13) bone marrow (BM) engraftment of human CD45/71 cells compared to non-expanded controls (18.6±12.5; n=5). When the –COOH expanded cells (n=5) (dose: 10x10⁷ cells/kg) were cotransplanted with non-expanded cells (dose: 2.5x10⁷ cells/kg) from the same UCB it gave better BM frequency of CD45/71 cells (23.8±13.1 %; p=0.31), CD34 progenitor (1.5±0.9%; p=0.29) and CD19/20 B-lineage cells (3.3±1.7%; p=0.32) compared to control cultures (7.2±3.7 %, 0.2±0.2 % & 1.1±0.7% respectively; n=4). NSG mice transplanted with expanded cells (±–COOH or ±non-expanded cells) had a significantly higher (p<0.05) survival rate (85.4%; n=48) compared to non-expanded controls (50.0%; n=12) due to lower incidences of graft-versus-host-disease.

In conclusion, surface functionalization of SWCNT scaffold plays a critical role in mediating viability, mitochondrial superoxide & subsequent expansion of freeze-thawed, non-enriched UCB-MNC with –COOH giving the optimal effect. Moreover, cotransplantation of low dose non-expanded cells along with high dose of –COOH expanded cells from the same cord unit boosted the engraftment kinetics of human cells with better survival rates in an immunodeficient mice model. Such studies may pave the development of immobilized carbon nanotubes films that could have the potential for clinical translation.