Valentina Tirelli

Humanized culture medium for clinical expansion of human erythroblasts.

Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) using media with serum previously defined as human erythroid massive amplification culture (HEMAser). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated a 6–12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10−6 M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMAser were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMAdef). HEMAdef sustained erythroid amplification as efficiently as HEMAser for cord blood MNC and 10-fold higher than HEMAser for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMAdef by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.

Under HEMA conditions, self-replication of human erythroblasts is limited by autophagic death☆

The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (~10(10)) are insufficient for transfusion (~10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.

Transcriptomic and phospho-proteomic analyzes of erythroblasts expanded in vitro from normal donors and from patients with polycythemia vera.

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV‐derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV‐derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic‐derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho‐profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV‐derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera. Am. J. Hematol. 88:723–729, 2013.

Phenotypic Definition of the Progenitor Cells with Erythroid Differentiation Potential Present in Human Adult Blood

In Human Erythroid Massive Amplification (HEMA) cultures, AB mononuclear cells (MNC) generate 1-log more erythroid cells (EBs) than the corresponding CD34pos cells, suggesting that MNC may also contain CD34neg HPC. To clarify the phenotype of AB HPC which generate EBs in these cultures, flow cytometric profiling for CD34/CD36 expression, followed by isolation and functional characterization (colony-forming-ability in semisolid-media and fold-increase in HEMA) were performed. Four populations with erythroid differentiation potential were identified: CD34posCD36neg (0.1%); CD34posCD36pos (barely detectable-0.1%); CD34negCD36low (2%) and CD34negCD36neg (75%). In semisolid-media, CD34posCD36neg cells generated BFU-E and CFU-GM (in a 1 : 1 ratio), CD34negCD36neg cells mostly BFU-E (87%) and CD34posCD36pos and CD34negCD36low cells were not tested due to low numbers. Under HEMA conditions, CD34posCD36neg, CD34posCD36pos, CD34negCD36low and CD34negCD36neg cells generated EBs with fold-increases of ≈9,000, 100, 60 and 1, respectively, and maturation times (day with >10% CD36highCD235ahigh cells) of 10–7 days. Pyrenocytes were generated only by CD34neg/CD36neg cells by day 15. These results confirm that the majority of HPC in AB express CD34 but identify additional CD34neg populations with erythroid differentiation potential which, based on differences in fold-increase and maturation times, may represent a hierarchy of HPC present in AB.

HIV-1 Nef impairs key functional activities in human macrophages through CD36 downregulation.

Monocytes and macrophages utilize the class A and B scavenger receptors to recognize and perform phagocytosis of invading microbes before a pathogen-specific immune response is generated. HIV-1 Nef protein affects the innate immune system impairing oxidative burst response and phagocytic capacity of macrophages. Our data show that exogenous recombinant myristoylated Nef protein induces a marked CD36 downregulation in monocytes from Peripheral Blood Mononuclear Cells, in Monocyte-Derived Macrophages (MDMs) differentiated by cytokines and in MDMs contained in a mixed culture obtained expanding PBMCs under Human Erythroid Massive Amplification condition. Under the latter culture condition we identify three main populations after 6 days of expansion: lymphocytes (37.8±14.7%), erythroblasts (46.7±6.1%) and MDMs (15.7±7.5%). The Nef addition to the cell culture significantly downregulates CD36 expression in MDMs, but not in erythroid cells. Furthermore, CD36 inhibition is highly specific since it does not modify the expression levels of other MDM markers such as CD14, CD11c, CD86, CD68, CD206, Toll-like Receptor 2 and Toll-like Receptor 4. Similar results were obtained in MDMs infected with VSV-G pseudotyped HIV-1-expressing Nef. The reduced CD36 membrane expression is associated with decrease of correspondent CD36 mRNA transcript. Furthermore, Nef-induced CD36 downregulation is linked to both impaired scavenger activity with reduced capability to take up oxidized lipoproteins and to significant decreased phagocytosis of fluorescent beads and GFP-expressing Salmonella tiphymurium. In addition we observed that Nef induces TNF-α release in MDMs. Although these data suggest a possible involvement of TNF-α in mediating Nef activity, our results exclude a possible relationship between Nef-induced TNF-α release and Nef-mediated CD36 downregulation. The present work shows that HIV-1 Nef protein may have a role in the strategies elaborated by HIV-1 to alter pathogen disease outcomes, by modulating CD36 expression in macrophages, favoring the onset of opportunistic infections in HIV-1 infected people.

Recovery and Biodistribution of Ex Vivo Expanded Human Erythroblasts Injected into NOD/SCID/IL2Rγ mice.

Ex vivo expanded erythroblasts (EBs) may serve as advanced transfusion products provided that lodgment occurs in the macrophage-niche of the marrow permitting maturation. EBs expanded from adult and cord blood expressed the receptors (CXCR4, VLA-4, and P-selectin ligand 1) necessary for interaction with macrophages. However, 4-days following transfusion to intact NOD/SCID/IL2Rγ(null) mice, CD235a(pos) EBs were observed inside CD235a(neg) splenic cells suggesting that they underwent phagocytosis. When splenectomized and intact NOD/SCID/IL2Rγ(null) mice were transfused using retrovirally labeled human EBs, human cells were visualized by bioluminescence imaging only in splenectomized animals. Four days after injection, human CD235a(pos) cells were detected in marrow and liver of splenectomized mice but only in spleen of controls. Human CD235a(pos) erythrocytes in blood remained low in all cases. These studies establish splenectomized NOD/SCID/IL2Rγ(null) mice as a suitable model for tracking and quantification of human EBs in vivo.

TRANSPLANTATION AND CELLULAR ENGINEERING: Long‐term storage does not alter functionality of in vitro generated human erythroblasts: implications for ex vivo generated erythroid transfusion products

BACKGROUND: Cultured human erythroid cells derived in vitro may represent alternative transfusion products. It is unknown, however, if these ex vivo expanded erythroid cells remain functional or develop genetic abnormalities after storage.

Mononuclear cells from a rare blood donor, after freezing under good manufacturing practice conditions, generate red blood cells that recapitulate the rare blood phenotype.

Cultured red blood cells (cRBCs) from cord blood (CB) have been proposed as transfusion products. Whether buffy coats discarded from blood donations (adult blood [AB]) may be used to generate cRBCs for transfusion has not been investigated.

TRANSPLANTATION AND CELLULAR ENGINEERING: Long-term storage does not alter functionality of in vitro generated human erythroblasts: implications for ex vivo generated erythroid transfusion products: LONG-TERM STORAGE OF HUMAN ERYTHROBLAST

BACKGROUND: Cultured human erythroid cells derived in vitro may represent alternative transfusion products. It is unknown, however, if these ex vivo expanded erythroid cells remain functional or develop genetic abnormalities after storage.