Expansion, differentiation, and culture of hematopoietic stem & progenitor cells using an ancillary GMP-compliant medium
Bibliographic record
Abstract
Abstract Hematopoietic stem and/or progenitor cells (HSPCs) from human primary or pluripotent stem cells (hPSCs) and their derivatives, natural killer (NK), B, and T cells, hold promise as effective cellular therapies to treat various diseases, including malignancies. A significant challenge in manufacturing HSPC-derived cellular therapies is culturing sufficient numbers of HSPCs while preserving their potency and self-renewal. Concurrently, ex vivo differentiation of HSPCs into immune cells offers a cost-effective, allogeneic source for immune cell therapy. However, this entire process relies on reliable cell culture reagents that can support the expansion of functional HSPCs, as well as their subsequent differentiation into various blood cells, including T and NK cells. For clinical-grade cell and gene therapy products, high-quality ancillary materials compliant with current good manufacturing practice (cGMP) regulations are a requirement. We developed a cGMP-compliant culture medium designed to support the maintenance, expansion, and differentiation of HSPCs from both human primary and hPSC-derived progenitors. This novel HSPC medium is serum- and phenol red-free, and specifically designed to support further cell therapy manufacturing of HSPCs and HSPC-derived T and NK cells. To evaluate the novel HSPC medium, CD34+ HSPCs were isolated immunomagnetically from cord blood (CB) and cultured in HSPC medium supplemented with StemSpan™ CD34+ Expansion Supplement containing Flt3L, SCF, TPO, IL-3, and IL-6 plus 1 μM UM729 for 7 days. Expanded CB-derived CD34+ cells showed an average fold expansion of 117 ± 21 cells per input HSPC, with 53 ± 5% retaining CD34 expression (n = 6; mean ± SEM) after 7 days of culture. Notably, 6 ± 1% of the cells were also CD34+CD45RA-CD90+EPCR+, a phenotype indicative of a hematopoietic stem cell-enriched population. The yield of CD34+CD45RA-CD90+EPCR+ cells was 8 ± 2 per input cell. T and NK Differentiation potential was assessed by using HSPC medium supplemented with commercially available reagents. For differentiation to CD4+CD8+ double-positive (DP) T cells, CB- and hPSC-derived CD34+ HSPCs were differentiated for 42 and 28 days, respectively. CB-derived cultures displayed an average frequency of 20 ± 8% CD4+CD8+ cells with a yield of 21,300 ± 16,800 cells per input CD34+ HSPC (n = 6), while hPSC-derived cultures generated 34 ± 14% to CD4+CD8+ cells with a yield of 31 ± 7 cells per input CD34+ cell (n = 3; mean ± SEM). Notably, 10 ± 2% of the CB-derived and 23 ± 8% of the hPSC-derived CD4+CD8+ DP T cells also expressed CD3 and TCRαß, indicating a more differentiated phenotype. DP T cells from both cell sources were able to differentiate to more mature CD8 single-positive T cells. For differentiation to CD56+ NK cells, CB- and hPSC-derived CD34+ HSPCs were differentiated for 28 and 21 days, respectively. CB-derived cultures displayed an average frequency of 89 ± 1% CD56+ cells with a yield of 20,300 ± 3,700 cells per input CD34+ HSPC (n = 6; mean ± SEM ), while hPSC-derived cultures generated 84 ± 3% to CD56+ cells with a yield of 464 ± 132 cells per input CD34+ cell (n = 13; mean ± SEM). These results highlight the utility of this HSPC medium to generate high yields of T and NK cells required for large-scale cell manufacturing. Taken together, our data show that this novel cGMP-compliant HSPC medium enables efficient cord blood- and hPSC-derived HSPC expansion and differentiation to T and NK cells—both of which are crucial to meet the demands of immunotherapy research. These results demonstrate its potential to facilitate the development of cell and gene therapies, where their successful clinical manufacturing relies on robust and scalable cell expansion and differentiation protocols.
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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) | 0.001 | 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".