AA2P-mediated DNA demethylation synergizes with stem cell agonists to promote expansion of hematopoietic stem cells.

Small molecules have enabled expansion of hematopoietic stem and progenitor cells (HSPCs), but limited knowledge is available on whether these agonists can act synergistically. In this work, we identify a stem cell agonist in AA2P and optimize a series of stem cell agonist cocktails (SCACs) to help promote robust expansion of human HSPCs. We find that SCACs provide strong growth-promoting activities while promoting retention and function of immature HSPC. We show that AA2P-mediated HSPC expansion is driven through DNA demethylation leading to enhanced expression of AXL and GAS6. Further, we demonstrate that GAS6 enhances the serial engraftment activity of HSPCs and show that the GAS6/AXL pathway is critical for robust HSPC expansion.

Dimethyl sulfoxide-free cryopreservation solutions for hematopoietic stem cell grafts.

BACKGROUND AIMS: The use of effective methods for the cryopreservation of hematopoietic stem cells (HSCs) is vital to retain the maximum engraftment activity of cord blood units (CBUs). Current protocols entail the use of dimethyl sulfoxide (DMSO) as intracellular cryoprotective agent (CPA) and dextran and plasma proteins as extracellular CPAs, but DMSO is known to be cytotoxic, and its infusion in patients is associated with mild to moderate side effects. However, new, commercially available, DMSO-free cryopreservation solutions have been developed, but their capacity to protect HSCs remains poorly investigated. METHODS: Herein the authors compared the capacity of four DMSO-free freezing media to cryopreserve cord blood (CB) HSCs: CryoProtectPureSTEM (CPP-STEM), CryoScarless (CSL), CryoNovo P24 (CN) and Pentaisomaltose (PIM). Clinical-grade DMSO/dextran solution was used as control. RESULTS: Of the four cryopreservation solutions tested, the best post-thaw cell viability, recovery of viable CD45+ and CD34+ cells and potency were achieved with CPP-STEM, which was equal or superior to that seen with the control DMSO. CSL provided the second best post-thaw results followed by PIM, whereas CN was associated with modest viability and potency. Further work with CPP-STEM revealed that CB CD34-enriched HSCs and progenitors cryopreserved with CPP-STEM maintained high viability and growth expansion activity. In line with this, a pilot transplantation assay confirmed that CPP-STEM-protected CB grafts supported normal short- and long-term engraftment kinetics. CONCLUSIONS: The authors' results suggest that new, valuable alternatives to DMSO are now available for the cryopreservation of HSCs and grafts, including CBUs.

Current and Future Perspectives for the Cryopreservation of Cord Blood Stem Cells.

Hematopoietic stem cell (HSC) transplantation is a well-established procedure for the treatment of many blood related malignancies and disorders. Before transplantation, HSC are collected and cryopreserved until use. The method of cryopreservation should preserve both the number and function of HSC and downstream progenitors responsible for long- and short-term engraftment, respectively. This is especially critical for cord blood grafts, since the cell number associated with this stem cell source is often limiting. Loss of function in cryopreserved cells occurs following cryoinjuries due to osmotic shock, dehydration, solution effects and mechanical damage from ice recrystallization during freezing and thawing. However, cryoinjuries can be reduced by 2 mitigation strategies; the use of cryoprotectants (CPAs) and use of control rate cooling. Currently, slow cooling is the most common method used for the cryopreservation of HSC graft. Moreover, dimethyl-sulfoxide (DMSO) and dextran are popular intracellular and extracellular CPAs used for HSC grafts, respectively. Yet, DMSO is toxic to cells and can cause significant side effects in stem cells' recipients. However, new CPAs and strategies are emerging that may soon replace DMSO. The aim of this review is to summarise key concepts in cryobiology and recent advances in the field of HSC cryobiology. Other important issues that need to be considered are also discussed such as transient warming events and thawing of HSC grafts.

Inhibition of ice recrystallization during cryopreservation of cord blood grafts improves platelet engraftment.

BACKGROUND: Platelet engraftment following cord blood (CB) transplantation remains a significant hurdle to this day. The uncontrolled growth of ice, a process referred to as ice recrystallization, is one of several mechanisms that lead to cell loss and decreased potency during freezing and thawing. We hypothesized that reducing cell damage induced by ice recrystallization in CB units (CBUs) would reduce losses of stem and progenitor cells and therefore improve engraftment. We previously demonstrated that the ice recrystallization inhibitor (IRI) N-(2-fluorophenyl)-D-gluconamide (IRI 2) increases the postthaw recovery of CB progenitors. Herein, we set out to ascertain whether IRI 2 can enhance platelet and bone marrow engraftment activity of hematopoietic stem cells (HSCs) in cryopreserved CBUs using a serial transplantation model. STUDY DESIGN AND METHODS: CBUs were processed following standard volume/red blood cell reduction procedure and portions frozen with dimethyl sulfoxide (DMSO) supplemented or not with IRI 2. Thawed CB samples were serially transplanted into immunodeficient mice. RESULTS: Our results show that supplementation of DMSO with IRI 2 had several beneficial effects. Specifically, higher levels of human platelets were observed in the peripheral blood (p < 0.05; n = 4) upon transplant of CBUs preserved with the IRIs. In addition, human BM chimerism and the number of human CFU progenitors in the bone marrow were superior in IRI 2 recipients compared to DMSO recipients. Moreover, IRI 2 had no negative impact on the multilineage differentiation and self-renewal activities of HSCs. DISCUSSION: Taken together, these results demonstrate that supplementation of a hematopoietic graft with IRI can improve the postthaw engraftment activities of HSCs.

Paracrine Factors Released by Osteoblasts Provide Strong Platelet Engraftment Properties.

Ex vivo expansion of hematopoietic stem cell (HSCs) and progenitors may one day overcome the slow platelet engraftment kinetics associated with umbilical cord blood transplantation. Serum-free medium conditioned with osteoblasts (i.e., osteoblast-conditioned medium [OCM]) derived from mesenchymal stromal cells (MSC) was previously shown to increase cell growth and raise the levels of human platelets in mice transplanted with OCM-expanded progenitors. Herein, we characterized the cellular and molecular mechanisms responsible for these osteoblast-derived properties. Limiting dilution transplantation assays revealed that osteoblasts secrete soluble factors that synergize with exogenously added cytokines to promote the production of progenitors with short-term platelet engraftment activities, and to a lesser extent with long-term platelet engraftment activities. OCM also modulated the expression repertoire of cell-surface receptors implicated in the trafficking of HSC and progenitors to the bone marrow. Furthermore, OCM contains growth factors with prosurvival and proliferation activities that synergized with stem cell factor. Insulin-like growth factor (IGF)-2 was found to be present at higher levels in OCM than in control medium conditioned with MSC. Inhibition of the IGF-1 receptor, which conveys IGF-2' intracellular signaling, largely abolished the growth-promoting activity of OCM on immature CD34+ subsets and progenitors in OCM cultures. Finally, IGF-1R effects appear to be mediated in part by the coactivator ß-catenin. In summary, these results provide new insights into the paracrine regulatory activities of osteoblasts on HSC, and how these can be used to modulate the engraftment properties of human HSC and progenitors expanded in culture. Stem Cells 2019;37:345-356.

Human Bone Marrow Mesenchymal Stromal Cell-Derived Osteoblasts Promote the Expansion of Hematopoietic Progenitors Through Beta-Catenin and Notch Signaling Pathways.

Coculture of hematopoietic stem cells (HSC) with primary stromal cells from HSC niches supports the maintenance and expansion of HSC and progenitors ex vivo. However, a major drawback is the availability of primary human samples for research and clinical applications. We investigated the use of in vitro derived osteoblasts as a new source of feeder cells and characterized the molecular pathways that mediate their growth-promoting activities. First, we compared the growth and differentiation modulating activities of mesenchymal stromal cells (MSC)-derived osteoblasts (M-OST) with those of their undifferentiated precursor on umbilical cord blood (UCB) progenitors. Feeder-free cultures were also included as baseline control. Cell growth and expansion of hematopoietic progenitors were significantly enhanced by both feeder cell types. However, progenitor cell growth was considerably greater with M-OST. Coculture also promoted the maintenance of immature CD34+ progenitor subsets and modulated in a positive fashion the expression of several homing-related cell surface receptors, in a feeder-specific fashion. Serial transplantation experiments revealed that M-OST coculture supported the maintenance of long-term lympho-myeloid reconstituting HSC that provided engraftment levels that were generally superior to those from MSC cocultures. Mechanistically, we found that coculture with M-OST was associated with enhanced beta-catenin (ß-Cat) activity in UCB cells and that abrogation of ß-Cat/T-cell factor activity blunted the growth-promoting activity of the M-OST coculture. Conversely, Notch inhibition reduced UCB cell expansion, but to a much lesser extent. In conclusion, this study demonstrates that M-OST are excellent feeder cells for HSC and progenitors, and it identifies key molecular pathways that are responsible for the growth-enhancing activities of osteoblasts on UCB progenitors.

Small-Molecule Ice Recrystallization Inhibitors Improve the Post-Thaw Function of Hematopoietic Stem and Progenitor Cells.

The success of hematopoietic stem cell transplantation depends in part on the number and the quality of cells transplanted. Cryoinjuries during freezing and thawing reduce the ability of hematopoietic stem and progenitor cells (HSPCs) to proliferate and differentiate after thawing. Up to 20% of the patients undergoing umbilical cord blood (UCB) transplant experience delayed or failed engraftment, likely because of the inadequate hematopoietic potency of the unit. Therefore, the optimization of cryopreservation protocols, with an emphasis on the preservation of HSPCs, is an important issue. Current protocols typically utilize a 10% dimethyl sulfoxide cryoprotectant solution. This solution ensures 70-80% post-thaw cell viability by diluting intracellular solutes and maintaining the cell volume during cryopreservation. However, this solution fails to fully protect HSPCs, resulting in the loss of potency. Therefore, a new class of cryoprotectants (N-aryl-d-aldonamides) was designed and assessed for the ability to inhibit ice recrystallization and to protect HSPCs against cryoinjury. Several highly active ice recrystallization inhibitors were discovered. When used as additives to the conventional cryoprotectant solution, these nontoxic small molecules improved the preservation of functionally divergent hematopoietic progenitors in the colony-forming unit and long-term culture-initiating cell assays. By contrast, structurally similar compounds that did not inhibit ice recrystallization failed to improve the post-thaw recovery of myeloid progenitors. Together, these results demonstrate that the supplementation of cryopreservation solution with compounds capable of controlling ice recrystallization increases the post-thaw function and potency of HSPCs in UCB. This increase may translate into reduced risk of engraftment failure and allow for greater use of cryopreserved cord blood units.