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.

Severe fetal anemia caused by anti-Jra : Burst forming unit-erythroid colony formation inhibition assay suggesting possible erythroid suppression mechanism.

BACKGROUND: The Jr blood group system includes a single, high-prevalence antigen, Jra , encoded by the ABCG2 gene. The impact of anti-Jra in pregnancy is variable, ranging from no clinical effect to severe anemia including some fetal deaths. Case reports have postulated that anti-Jra mediated fetal anemia is poorly hemolytic, suggesting other mechanisms of anemia may be involved. STUDY DESIGN AND METHODS: We describe the case of severe anti-Jra mediated fetal anemia. At Canadian Blood Services laboratories, maternal anti-Jra was tested for phagocytic activity via a monocyte monolayer assay (MMA) and erythroid suppression via inhibition of burst forming unit-erythroid (BFU-E) colony formation assays. The New York Blood Center sequenced exons 4 and 7 of the ABCG2 gene. RESULTS AND DISCUSSION: Sequencing of exons 4 and 7 of the ABCG2 gene revealed maternal compound heterozygosity for two nonsense mutations at exon 7 (c.706 C > T and c.784G > T). Fetal sequencing revealed the c.706C > T polymorphism. The MMA showed a borderline phagocytic index (around the cutoff of five for both donor segments tested [5 ± 1 and 7 ± 3]). The BFU-E colony formation inhibition assay suggested a dose-dependent inhibition of BFU-E colony formation with inhibition percentages of 4%, 11%, and 43% at maternal serum concentrations of 2%, 5%, and 10%, respectively. Our findings support the hypothesis that anti-Jra may impair erythropoiesis leading to clinically significant fetal/neonatal anemia. A referral to maternal fetal medicine is recommended if anti-Jra is detected in pregnancy, regardless of the titer.

Contamination of platelet concentrates with Staphylococcus aureus induces significant modulations in platelet functionality.

BACKGROUND AND OBJECTIVES: Platelet concentrates (PCs) contaminated with Staphylococcus aureus can escape detection during PC screening, causing septic transfusion reactions. This study aimed to determine the impact of S. aureus contamination on platelet metabolism and functionality during PC storage. MATERIALS AND METHODS: Targeted metabolomics (N = 3) was performed on non-spiked PCs and PCs inoculated with 10-20 colony-forming units (CFU)/bag of S. aureus. Metabolites were quantified at 0, 48 and 144 h using high-performance mass spectrometry (MS). Additionally, PCs spiked with approximately 20 CFU/bag of S. aureus were sampled every 24 h for up to 144 h to evaluate platelet functionality using flow cytometry (N = 2). RESULTS: Eight metabolites had significantly different levels in spiked PCs (log2 fold-change ≤ or ≥±1) versus non-spiked units at 48 and 144 h. Xanthine, uridine, serine, glutamine and threonine were increased, whereas orotic acid, dihydroorotic acid and aspartic acid were decreased. Flow cytometry showed a significant decrease in expression of GPIIb while P-selectin expression was significantly increased in spiked PCs after 72 h of storage when S. aureus concentration was ≥10E+08 CFU/ml. Additionally, phosphatidylserine exposure was significantly increased after 48 h of PC storage, when S. aureus had reached a concentration of 2E+06. CONCLUSION: Contamination with S. aureus exacerbates platelet storage lesions in contaminated PCs but only when the bacterium has reached clinically significant levels.

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.

Insights into the Hematopoietic Regulatory Activities of Osteoblast by Secretomics.

Osteoblasts are a key component of the endosteal hematopoietic stem cell (HSC) niche and are recognized with strong hematopoietic supporting activity. Similarly, mesenchymal stromal cells (MSC)-derived osteoblast (M-OST) conditioned media (OCM) enhances the growth of hematopoietic progenitors in culture and modulate their engraftment activity. We aimed to characterize the hematopoietic supporting activity of OCM by comparing the secretome of M-OST to that of their precursor. Over 300 proteins were quantified by mass spectroscopy in media conditioned with MSC or M-OST, with 47 being differentially expressed. Included were growth factors, extracellular matrix (ECM) proteins and proteins from the complement pathways. The functional contribution of selected proteins on the growth and differentiation of cord blood (CB) progenitors was tested. Secreted Protein Acidic and Rich in Cysteine (SPARC) and Galectin 3 (Gal3) had little impact on the growth of CB cells in serum-free medium (SFM). In contrast, inhibition of the complement 3 A receptor (C3a-R) present on CB progenitors significantly reduced the growth of CD34+ cells in OCM cultures but not in SFM. These results provide new insights into changes in factors released by MSC undergoing osteoblast differentiation, and on paracrine factors that are partially responsible for the hematopoietic supporting activity of osteoblasts. This article is protected by copyright. All rights reserved.

Transient warming affects potency of cryopreserved cord blood units.

BACKGROUND AIMS: Cryopreserved cord blood units (CBUs) can be exposed to transient warming events (TWEs) during routine banking operations, which may affect their potency. NetCord-FACT guidelines recommend removal of these CBUs from inventory. The objective of this work was to evaluate warming kinetics of frozen CBUs in different settings to determine the optimal working environment and define the impact of different TWE scenarios on CB post-thaw quality and potency. METHODS: The warming kinetics of frozen CBUs was influenced by both working surfaces and ambient working temperature, with cold plates providing better protection than vinyl or metal surfaces. Measurement of time for required operational activities revealed that CBUs are probably exposed to core temperatures greater than -150°C even when cold plates are used to reduce warming rates. RESULTS: On the basis of the warming kinetics and observed operational activities, three TWE causing scenarios (control, typical, worst case) were investigated using a pool-and-split design and cell viability, recovery and potency (colony-forming unit [CFU]) assays were performed. TWEs were found to have little impact on the recovery of total nucleated cells or on the viability of CD34+ cells. In contrast, the viability and recovery of CD45+ cells in the smaller CBU compartments were reduced by TWEs. Moreover, the worst-case TWE reduced CFU recovery from CBUs, whereas the typical-scenario TWE had little effect. CONCLUSIONS: Our results demonstrate that the distal segment underestimates the viability and potency of CBUs and that TWEs can affect the post-thaw viability and potency of CBUs. Although TWEs are almost inevitable during cord-blood banking operations, their effects must be diminished by reducing exposure time, using cold plates and strict operational protocols, to prevent worst-case TWEs.

Overcoming the deceptively low viability of CD45+ cells in thawed cord blood unit segments.

BACKGROUND AND OBJECTIVES: There is no standard methodology for post-thaw sample preparation for viability analysis of umbilical cord blood units (CBU). A common challenge faced by CB bank is for their product to meet the post-thaw cell viability threshold for CD45+ cells set at 40% by NetCord-FACT. The objective of this work was to improve the post-thaw staining method to maximize CD45+ cell viability so that clinically valuable samples meet the NetCord-FACT threshold criteria for CD45+ and CD34+ cell viabilities. MATERIALS AND METHODS: Samples of CBU buffy coats and CBU segments were thawed and taken for staining. Various parameters were evaluated on CD45+ and CD34+ cell viability as measured by 7-actinomycin D (7-AAD) staining. RESULTS: The results revealed that initiating the staining at 20 min post-thaw instead of 30, shortening the red cell lysis treatment, or performing lysis on ice and removing this step all together, all improved the viability of CD45+ cells. Using CBU segments, it was shown that the most effective approach in increasing the viability of CD45+ cells was the complete omission of red cell lysis step. However, removal of the lysis step can create technical artefacts during flow cytometry acquisition that results in an underestimation of the viability of CD34+ cells. This can be avoided and CD34+ cell viability restored with additional thresholding on CD45 signal. CONCLUSION: CB CD45+ cells are sensitive to red cell lysis treatment post-thaw; omission of this step provides the best viability and ultimately better reflects the quality of cells used for transplantation.

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.

Development and testing of a stepwise thaw and dilute protocol for cryopreserved umbilical cord blood units.

BACKGROUND: It is clinically important to maintain high viability and potency of umbilical cord blood units (CBUs) for transplantation during thawing. In the absence of a standard thawing protocol, this study was designed to develop one based on the consensus practice of transplant centers and address the shortage of dextran 40 thawing solution. STUDY DESIGN AND METHODS: Frozen CBU aliquots were thawed using dextran 40 thawing solution while manipulating temperature and volume of diluent and mode of dilution. The effects of these on CD45+ and CD34+ cell viability were measured through annexin V and SYTOX green staining. The developed protocol was then used to compare dextran 40 and PLASMA-LYTE A thawing solutions and finally tested on whole CBUs. RESULTS: Step-by-step investigations resulted in the development of a protocol that thaws and dilutes CBUs with room temperature diluent to five times the original volume using two sequential dilutions separated by equilibration times. PLASMA-LYTE A diluent provided superior viability of CD45+ and CD34+ cells than dextran 40 and recovered more colony-forming units. However, both diluents were equally effective in maintaining stability of the thawed CBU for 4 hours. Moreover, the stem cell-enriched CD34+CD38- subpopulations appeared more resistant to cryoinjuries than their CD34+CD38+ counterpart. CONCLUSION: The developed thawing protocol recovers viable CD45+ and CD34+ cells above the standard thresholds and maintains CBU potency. PLASMA-LYTE A for thawing solution proved to be an efficient alternative to dextran 40. Finally, greater dilution should be avoided to maintain the viability of CD45+ cells and maximize graft cell dose.

Impact of osteoblast maturation on their paracrine growth enhancing activity on cord blood progenitors.

BACKGROUND: Osteoblasts possess strong growth modulatory activity on haematopoietic stem cells and progenitors. We sought to characterise the growth and differentiation modulatory activities of human osteoblasts at distinct stages of maturation on cord blood (CB) progenitors in the context of osteoblast conditioned medium (OCM). METHODS: OCM was produced from MSC-derived osteoblasts (M-OST) at distinct stages of maturation. The growth modulatory activities of the OCM were tested on CB CD34+ cells using different functional assays. RESULTS: OCMs raised the growth of CB cells and expansion of CD34+ cells independently of the maturation status of M-OST. However, productions of immature CB cells including committed and multipotent progenitors were superior with OCM produced with immature osteoblasts. Osteogenic differentiation was accompanied by the upregulation of IGFBP-2, by several members of the Angpt-L family of growth factor, and by the Notch ligands Dll-1 and Dll-4. However, the growth activity of OCM and the in vivo engraftment properties of OCM-expanded CB cells were retained after IGFBP-2 neutralisation. Similarly, OCM-mediated expansion of CB myeloid progenitors was largely independent of Notch signalling. CONCLUSIONS: These results demonstrate that immature osteoblasts possess greater regulatory activity over haematopoietic progenitors, and that this activity is not entirely dependent on Notch signalling.

Characterization of the growth modulatory activities of osteoblast conditioned media on cord blood progenitor cells.

Engraftment outcomes are strongly correlated with the numbers of hematopoietic stem and progenitor cells (HSPC) infused. Expansion of umbilical cord blood (CB) HSPC has gained much interest lately since infusion of expanded HSPC can accelerate engraftment and improve clinical outcomes. Many novel protocols based on different expansion strategies of HSPC and their downstream derivatives are under development. Herein, we describe the production and properties of serum-free medium (SFM) conditioned with mesenchymal stromal cells derived-osteoblasts (OCM) for the expansion of umbilical CB cells and progenitors. After optimization of the conditioning length, we show that OCM increased the production of human CB total nucleated cells and CD34+ cells by 1.8-fold and 1.5-fold over standard SFM, respectively. Production of immature CD34+ subpopulations enriched in hematopoietic stem cells was also improved with a shorter conditioning period. Moreover, we show that the growth modulatory activities of OCM on progenitor expansion are regulated by both soluble factors and non-soluble cellular elements. Finally, the growth and differentiation modulatory activities of OCM were fully retained after high dose-ionizing irradiation and highly stable when OCM is stored frozen. In summary, our results suggest that OCM efficiently mimics some of the natural regulatory activities of osteoblasts on HSPC and highlight the marked expansion potentials of SFM conditioned with osteoblasts.

Medium conditioned with mesenchymal stromal cell-derived osteoblasts improves the expansion and engraftment properties of cord blood progenitors.

Strategies to enhance the expansion of umbilical cord blood hematopoietic stem and progenitor cells (HSPCs) are crucial to enable their widespread application to adults and to overcome important limitations, such as delayed engraftment. Osteoblasts regulate HSPCs under steady-state and also under stress conditions, when HSPCs undergo numerous cycles of expansion. We hypothesized that osteoblasts could provide better stimulation for the expansion of multipotent HSPCs and subsequent hematopoietic recovery than mesenchymal stromal cells. Hence, we assessed the growth and engraftment modulatory activities of mesenchymal stromal cell-derived osteoblasts (M-OSTs) on hematopoietic progenitors. Mesenchymal stromal cells and M-OSTs favored the maintenance of CD34(+) cells. The expansion of cord blood CD34(+) cells and myeloid progenitors was highest in cultures supplemented with unfiltered M-OST-conditioned medium (M-OST CM). In addition, increased expression of cell surface receptors important for the homing of progenitors to the bone marrow, C-X-C chemokine receptor type 4 and lymphocyte function-associated antigen 1, was observed in CM-based cultures. Additionally, M-OST CM positively modulated the engraftment properties of expanded progenitors. Most notably, although human platelet levels remained steady in the first 2 weeks in mice transplanted with HSPCs expanded in standard medium, levels in mice transplanted with M-OST CM HSPCs rose continuously. Consistent with this, short-term human progenitor reconstitution was consistently greater in M-OST recipients. Finally, cytokine array-based profiling revealed increases in insulin-like growth factor binding protein 2, chemokines, and myeloid stimulating cytokines in M-OST CM. In conclusion, this study suggests that M-OSTs represent a new underappreciated source of feeder cells for the expansion of HSPCs with enhanced thrombopoietic activity.

Lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells.

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments. Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Immunologic basis and immunoprophylaxis of RhD induced hemolytic disease of the newborn (HDN).

RhD antigen is the most immunogenic and clinically significant antigen of red blood cells after ABO system. It has historically been associated with hemolytic disease of the newborn (HDN) which is now routinely prevented by the administration of polyclonal anti-D immunoglobulin. This management of HDN has proven to be one of the most successful cases of prophylactic treatment based on antibody mediated immune suppression (AMIS). Despite the increasing efficiency of treatment, the mechanism of action of anti-D is not completely defined. There is a widespread interest in obtaining a reliable therapeutic monoclonal anti-D, due to difficulty of maintaining a pool of high titer volunteer donors for plasma collection and also increasing demand for antenatal prophylaxis and safety issues with plasma derived products. Candidate monoclonal anti-D preparations should demonstrate appropriate functionality in both in vitro and in vivo assays comparable to polyclonal anti-D immunoglobulin. These criteria are reviewed in addition to the factors regulating development of D specific immune response in D negative individuals and its suppression in HDN prophylaxis.

Specificity and isotype of Rh specific antibodies produced by human B-cell lines established from alloimmunized Rh negative women.

Despite the successful outcome of anti-D prophylaxis program, alloimmunization still occurs. The aim of this study was to examine the specificity and isotype of anti-Rh antibodies in plasma samples of Rh negative alloimmunized individuals and to study the same parameters in lymphoblastoid cell lines (LCLs) generated from the same donors. Specificity of anti-Rh antibodies was determined in plasma of nine alloimmunized subjects by direct hemagglutination using a panel of known RBC genotypes and isotype of specific antibodies were identified by an antigen specific ELISA. Similar methods were employed to determine specificity and isotype of antibodies produced by Rh specific LCLs established from four donors. LCLs were generated by Epstein-Barr virus transformation of peripheral blood mononuclear cells isolated from each donor followed by their culture over a feeder of human fetal fibroblasts. Upon emergence of lymphoblastoid cells, culture supernatants were assayed for presence of Rh specific antibody by hemagglutination assay. Anti-D was the predominant antibody in both plasma samples and among the 128 established LCLs; however, antibodies to other Rh specificities namely C and E were also produced. The isotype of anti-Rh antibody in all plasma samples was found to be IgG, predominantly IgG1, combined in 7 samples with IgM. Similarly 76%, 9.2% and 14.8% of LCLs were determined to produce antibody of IgG, IgM and of both isotypes, respectively. The data supported that the D antigen is the immunodominant component of the Rh system as indicated by the in vitro and in vivo profiles of Rh specificities in our alloimmunized subjects.