Insights into highly engraftable hematopoietic cells from 27-year cryopreserved umbilical cord blood

Protocol for enrichment and functional analysis of human hematopoietic cells from umbilical cord blood

“…There are a wide variety of fluorophore combinations that are possible, and this is one that has been validated and used in published studies. 1 , 8 , 9 , 11 , 12 There are also additional immunophenotypic markers of HSCs/HPCs that are not listed here but can be found in other studies. 12 , 13 , 14 For catalog numbers, refer to key resources table .…”

“…This can include adding fresh media (containing 2× concentration of cytokines) on top of existing media or centrifuging and resuspending cells in fresh media (containing 1× concentration of cytokines) and replating. 1 …”

“…We have utilized this protocol to determine the potency of unmanipulated cord blood units ( CBUs ), most recently to show that long-term cryopreserved CBUs maintain highly functional HSCs/HPCs. 1 Determining the potency of umbilical CB is crucial to its use, as there is a higher potential for graft failure following use of CB for hematopoietic cell therapies compared to other donor sources. 2 , 3 , 4 , 5 Understanding factors that influence potency may therefore lead to improved methods of collection, processing, or treatment to enhance transplantation outcomes.…”

“…While immunophenotyping and CFU assays are valuable tools, we have previously shown that there are specific instances when immunophenotypic numbers of HSCs/HPCs do not correlate well with in vivo engraftment 20 and CFU assays are sensitive to additional factors such as cryopreservation. 1 , 21 Expansion assays are valuable metrics of hematopoietic cell proliferation, but it is well established that HSCs/HPCs undergo biochemical and functional changes while growing in ex vivo culture systems. Finally, NSG mice, while a gold standard for modeling transplantation outcomes in the lab, do not harbor the same hematopoietic niches or factors as humans do.…”

The fulfilled promise and unmet potential of umbilical cord blood

“…For instance, a single center retrospective study demonstrated that closer HLA matching had a stronger effect in improving pediatric transplantations than did overall cell dose, suggesting that HLA matching should be prioritized over a certain threshold of cell dose [26 ▪ ]. Additionally, in the only functional study of long-term (>25 years) cryopreserved CB to date, Broxmeyer et al [27 ▪▪ ] demonstrated that CBUs stored for more than 27 years maintain similar numbers of stem and progenitor cells capable of engrafting in vivo compared to recently cryopreserved and fresh units. Indeed, a retrospective single center study supported this idea by showing similar levels of predicted potency for CBU stored for 29 years compared to CBU used successfully in patient transplantations [28].…”