Ex Vivo Expansion of Human SCID-Repopulating Cells Using Recombinant TAT-HOXB4 Protein.
Bibliographic record
Abstract
Abstract One of the major obstacles to the successful clinical application of hematopoietic stem cell (HSC) transplantation, particularly in the context of related haplotype-mismatched transplantation, unrelated cord blood transplants for adults, and grafts that are processed ex vivo to remove malignant, or alloreactive T cells, is the number of available long-term repopulating HSCs. The addition of soluble recombinant TAT-HOXB4 protein was recently reported to enable rapid in vitro expansion of murine HSCs that retain their in vivo proliferation and differentiation capacity. However, the ability of this recombinant protein to effectively expand human hematopoietic stem cells remains hypothetical. In addition, limited information is available on underlying mechanisms of HOXB4 HSC expansion. First, to determine the capacity of recombinant TAT-HOXB4 protein to promote human HSC expansion, we treated human CD34+ cells for 4 and 8 days with 40 nM, or 80 nM TAT-HOXB4 protein in X-Vivo 15 medium supplemented with Stem Cell Factor, TPO, IL-6 and Flt3-ligand. Cultures exposed to TAT-HOXB4 treatment for 8 days had no pronounced effect on the total cell yield. During this period, a 2-fold net loss of CFU-GEMM was observed for controls, in comparison to ~8-fold and ~5-fold expansions in response to 40 nM and 80 nM TAT-HOXB4 (p<0.05), respectively. Recombinant TAT-HOXB4 also induced ~10–15-fold expansion of large CFU-GM, compared to only ~2.5-fold increase for controls (p<0.05). HSC numbers were enumerated at the beginning and at the end of the treatment using the principle of limiting dilution in a 4-month NOD/SCID repopulation assay. Culture for 8 days in cytokines devoid of TAT-HOXB4 resulted in ~2-fold loss of SCID Repopulating Cells (SRCs), while cultures supplemented with 40 and 80 nM TAT-HOXB4 protein showed a 2.5-fold (95% CI 1.7 – 3.3 fold) and 5.5-fold (95% CI 3.6 – 7.4 fold) increase, respectively. Then, to determine whether the increase in HSC numbers resulted from HOXB4-enhanced proliferation of HSCs, we examined the cell cycle profile of control and HOXB4-treated cell populations using Hoechst 33342 and pyronin Y dyes. After 4-day treatment with 80 nM TAT-HOXB4, 44%±12% of CD34+CD38+ cells were in Go, compared to only 19%±6% of the controls (p<0.05). In contrast, similar proportions (89–91%) of quiescent CD34+CD38− cells were observed for both conditions. Tracking cell divisions using CFSE also showed that during this period, HOXB4-treated CD34+CD38+ cells underwent ~2 population doublings less than controls (p<0.05). In conclusion, short-term exposure of human CD34+ populations to recombinant TAT-HOXB4 protein has the potential to achieve clinically relevant HSC expansion levels. At the HSC level, these observations suggest that TAT-HOXB4 preferentially affects cell fate (self-renewal?) rather than cell proliferation.
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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 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.001 |
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".