Expansion Culture Of Hematopoietic Stem & Progenitor Cells From Frozen-Thawed, Non-Enriched Human Umbilical Cord Blood In Animal Component– & Serum–Free Medium Enhances Engraftment & Reduces Graft-Versus-Host-Disease
Bibliographic record
Abstract
Hematopoietic stem cell transplantation in adults using umbilical cord blood (UCB) is limited by low cell dosage & post-thaw viability. In several clinical trials cytokine supplementation & stromal cell support have been shown to enhance total nucleated cells (TNC). However, clinical safety is compromised due to source inconsistency & population heterogeneity of stromal cells along with animal components of the conventional growth media. In this study, we demonstrate effective use of an animal component– & serum–free growth medium to enhance the viability & ex vivo expansion of SCID repopulating cells (SRC) from frozen-thawed, non-enriched UCB–mononucleated cells (UCB-MNC). UCB-MNC were cultured in a commercially available animal component– & serum–free medium, StemSpanTM–ACF (ACF), while StemSpanTM–SFEM (SFEM), a conventional serum–free medium with human and bovine components served as control. Both media (from STEMCELL Technologies INC. Vancouver, Canada) were supplemented with clinical grade SCF, Flt-3 ligand, TPO, & IGFBP2. The expansion effects were characterized based on cell viability, phenotypic stem & progenitor cells & functional in vitro & in vivo assays. After 3-days of culturing, viability of CD45+ UCB-MNC was maintained at a significantly higher level in ACF (90.7±0.2%) compared to SFEM (75.4±0.1%) (p<0.0001; n=3). Culturing for 11-days significantly (p<0.0001; n=6) increased CD45+CD34+CD38– hematopoietic progenitors in ACF (90.6±13.5 fold) compared to control (4.8±0.4 fold). Further phenotypic study of ACF expanded cells showed significant increases of 4.1-fold for CD45+CD34+C38–CD90+ stem cells (p<0.0001), 2.1-fold for CD45+CD34+CD13+CD33+ myeloid progenitors (p<0.01) and 2.3-fold for CD45+CD34+C38–CD7+(p<0.01) lymphoid progenitors compared to SFEM (n=6). Viable TNC expansions were 4.3±0.2 fold and 5.9±0.7 fold in ACF and SFEM respectively (n=6; p<0.05). Colony forming unit (CFU) assay showed that ACF supported significantly higher expansion of GM progenitors than SFEM (60.1±7.9 vs. 14.6±2.1 fold; p<0.00001; n=16). The numbers of multi-potent progenitors, CFU-GEMM, were maintained in ACF but decreased in SFEM (0.83±0.21 vs. 0.09±0.04 fold relative to non-expanded UCB; p<0.01; n=16). UCB-MNC cultured for 11 days reconstituted the bone marrow (BM) of sub-lethally irradiated NOD/SCID gamma (NSG) mice with human CD45+/71+ cells as measured 16 weeks after transplantation at a dosage of 1x108 cells/kg. The frequency of human cells was higher for UCB expanded in ACF (38.1±15.4%; n=5) than for UCB expanded in SFEM (3.4±2.1; n=14; p<0.01). Human CD34+ progenitors were also detected in BM of the engrafted mice at frequencies of 2.4±1.4% and 0.2±0.1% for ACF and SFEM expanded cells respectively (p<0.05). Human hematopoiesis was multi-lineage with significantly higher numbers of CD45+/71+ & CD15+/66b+ granulopoietic cells (71.4-fold; p<0.001) and CD19+/20+ B-lineage cells (23.1-fold; p<0.001) in mice transplanted with cells expanded in ACF (n=5) as compared to SFEM (n=14). At a transplantation dosage of 2.5x107 cells/kg, non-expanded grafts (n=10) had similar engraftment of CD45+/71+cells compared to ACF expanded grafts (n=5; p=0.14), while engraftment was lower for SFEM expanded grafts (n=12; p<0.01). Limiting dilution analysis revealed that SRC frequencies were increased, on average, 7.9– and 1.2–fold in ACF relative to SFEM expanded & non-expanded grafts respectively. NSG mice transplanted with non-expanded grafts had a significantly lower (p<0.001) survival rate (40.4%, n=47) compared to those transplanted with grafts expanded in ACF (90.9%, n=11) or SFEM (92.3% n=26), or injected with saline only (100%, n=7). The high mortality rate in recipients of non-expanded grafts was due to higher incidence of graft-versus-host-disease (GVHD) associated with significantly (p<0.01; n=6) higher CD45+CD7+T cells in comparison to expanded grafts. In conclusion, expansion of freeze-thawed, non-enriched UCB-MNC in animal component– & serum–free medium improves in vivo repopulation and reduces mortality due to GVHD in a xenotransplantation model. These findings could set the platform for developing safer, cheaper & time efficient clinical transplantation, since no animal components, in the form of serum albumin or stromal cells, are required to achieve desired ex vivo expansion of hematopoietic stem & progenitor cells & pre-clinical outcomes. Disclosures: No relevant conflicts of interest to declare.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".