A recent study, published in Nature Medicine a week ago, proved the principal that hematopoietic stem/progenitor cells from cord blood (CB) unit could be successfully expanded for clinical use. Besides general excitement about these clinical results, I’d like to highlight that this study provided us a clinically-grade algorithm for validation of any new CB hematopoietic stem cell (HSC) expansion protocol.
I picked the most important points from the study and made up step-by-step protocol that I’m sharing them with you here. So, If you work on a new protocol for HSC expansion for potential clinical use, you should follow all steps, described below:
1. Optimize of in vitro expansion cell culture parameters: plastic, cytokines, serum-free expansion media, cell density, re-feeding…
2. Evaluate of HSC (CD34+/CD38-) or progenitors (CD34+ total) purity by flow cytometry and total cell number – before and after expansion.
3. Count fold expansion in vitro based on expression HSC surface markers by flow cytometry (at least CD34+/CD38- cells or even further) and determine optimal duration of expansion.
Engraftment of human HSC in the study was evaluated as more than 0.5% positive bone marrow cells for human CD45 at least 9 weeks after transplantation. Reconstitution should be multilineage (at least myeloid and lymphoid).
4. Do functionality test in vitro – colony assay (not shown in the study)
5. Assess of in vivo repopulation ability in xenotransplant models (NOD/SCID; NOG).
Groups – expanded, noncultured, fresh cells.
6. Calculate frequency of expanded HSC in vivo – limiting dilution transplantation assay.
7. Evaluate of self-renewal function of long-term expanded HSC in serial xenotransplant models.
8. Validate the reproducibility of protocols in cGMP conditions. Toxicology tests.
9. Apply for clinical trial.