Temporal Differences in Terminal Erythroid Differentiation of Human CD34+ Cells Derived From Cord Blood and Adult Peripheral Blood

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3200-3200
Author(s):  
Julie Jaffray ◽  
Jingping Hu ◽  
Jie Li ◽  
Xiuli An ◽  
Mohandas Narla
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Blood Cultures ◽  
Surface Markers ◽  
Cd34 Cells ◽  
Terminal Erythroid Differentiation ◽  
Kinetics Of ◽  
Adult Peripheral Blood

Abstract Abstract 3200 Purified CD34+ cells derived from either cord blood (CB) or peripheral blood (PB) are currently being used to further our molecular and mechanistic understanding of human terminal erythroid differentiation. What is unclear is whether there are differences in the kinetics of terminal erythroid differentiation of CD34+ cells from these two sources. In the present study, we document that terminal differentiation in cultured CD34+ cells purified from peripheral blood is faster than that of CD34+ cells from cord blood. For these studies, we optimized an 18 day, three phase, in vitro culture system using CD34+ cells to obtain enucleated reticulocytes. In this system, proerythroblasts are generated starting at day 6 which further differentiate during the duration of culture to eventually generate reticulocytes. Based on the expression of various membrane surface markers, we used flow cytometry to quantitatively monitor terminal erythroid differentiation from proerythroblasts to enucleated reticulocytes during culture. The three surface markers, alpha-4 integrin, band 3 and CD36 enabled us to clearly distinguish between all distinct stages of terminal erythroid differentiation – proerythroblasts, early- and late- basophilic erythroblasts, polychromatic and orthochromatic erythroblasts. These analyses enabled us to show that CD34+ cells purified and cultured from peripheral blood underwent terminal erythroid differentiation at a faster rate than CD34+ cells from cord blood. Terminal erythroid differentiation in cord blood cultures was delayed on an average of 2 to 3 days compared to peripheral blood. For example, the surface protein expression pattern seen on days 11–12 of cell culture of peripheral CD34+ cells was not achieved in cord blood cultures until day 14. This delay in terminal differentiation was also reflected by increased extents of enucleation in peripheral blood cultures compared to cord blood (culture day 12: 33% enucleation in PB and 7% in CB and on day 14: 45% enucleation in PB and 19% in CB). These findings have enabled us to document significant differences between the kinetics of terminal erythroid differentiation of CD34+ cells derived from fetal cord and adult peripheral blood. While at the present time we do not have a mechanistic understanding for this difference, we are currently exploring if the observed differences may be related to differences in cell cycle dynamics between fetal and adult erythropopiesis. Disclosures: No relevant conflicts of interest to declare.

Pomalidomide Transcriptionally Reprograms Adult Erythroid Progenitors Independently of Ikaros Proteasomal Degradation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 160-160
Author(s):  
Brian M. Dulmovits ◽  
Abena O. Appiah-Kubi ◽  
Julien Papoin ◽  
John Hale ◽  
Mingzhu He ◽  
...  
Keyword(s):  
Cord Blood ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Advisory Committees ◽  
Cd34 Cells ◽  
Terminal Erythroid Differentiation ◽  
Adult Peripheral Blood

Abstract Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. Disclosures Allen: Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Recombinant Tpo Stimulates Maturation of Megakaryocytes Derived from Adult Peripheral Blood- but Not Cord Blood-CD34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1191-1191
Author(s):  
Karen M. Pastos ◽  
William B. Slayton ◽  
Lisa M. Rimsza ◽  
Martha C. Sola
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Developmental Differences ◽  
Ploidy Levels ◽  
Cd34 Cells ◽  
Cord Blood Cd34 ◽  
Adult Bone ◽  
Adult Peripheral Blood ◽  
Cells Cultured

Abstract Umbilical cord blood (CB) is a valuable source of stem cells for transplantation. However, platelet engraftment is slow, taking approximately 70 days for CB transplants versus 20 days for mobilized adult peripheral blood (PB) transplants. This time is not significantly shortened by the administration of recombinant thrombopoietin (rTpo). The cause for the delay in platelet engraftment following CB transplant is unknown. We hypothesized that developmental differences in size and ploidy of neonatal versus adult megakaryocytes (MKs) contribute to this delay. To mimic these two types of transplant in vitro, we compared CB to PB CD34+ cells cultured in adult bone marrow stromal-conditioned media (CM) or unconditioned media (UCM) for 14 days. Increasing doses of rTpo were added to the CM, and the resulting MK maturation was compared with that of UCM with maximal rTpo concentration. MK number and ploidy were determined by flow cytometry using CD41-FITC and propidium iodide, respectively. Increased ploidy levels were expressed as percentage of MKs with a ploidy ≥ 8N. Results represent an average of three independent experiments. Figure Figure As seen in the figure, PB-derived MKs reached highest ploidy levels in the presence of UCM + 100 ng/ml rTpo. When cultured in CM, they exhibited lower ploidy levels, regardless of Tpo concentration. In contrast, CB-derived MKs exhibited higher ploidy levels in response to CM with either 0 or 0.1 ng/ml (physiologic concentration) of rTpo, as compared to higher rTpo concentrations or UCM + 100 ng/ml rTpo. MK numbers increased in response to rTpo in a dose-response manner, regardless of the source of the MKs (data not shown). These results indicate that intrinsic differences between CB- and PB-derived megakaryocytes exist, and that maturation is regulated differently in neonatal versus adult MKs. While Tpo is a potent stimulator of MK maturation in PB-derived MKs, it appears to inhibit this process in CB-derived MKs. These differences may be relevant to understanding the delayed platelet engraftment following CB transplants.

HMGB1 Is a Key Modulator Of Stress Erythropoiesis During Sepsis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 8-8 ◽  
Author(s):  
Sergio I Valdés-Ferrer ◽  
Lionel Blanc ◽  
Sebastien Didier ◽  
Johnson M. Liu ◽  
Jeffrey M. Lipton ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Cord Blood ◽  
Critical Role ◽  
Cecal Ligation ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Stress Erythropoiesis ◽  
Cd34 Cells ◽  
Sepsis Survivors ◽  
Terminal Erythroid Differentiation

Abstract Severe sepsis is a leading cause of death and disability. Anemia in sepsis survivors affects close to 100% of patients after the third day of in-hospital stay, regardless of blood levels on admission. Circulating levels of Erythropoietin (Epo) are low; paradoxically, administration of recombinant Epo is ineffective, and related to increased morbidity. During sepsis, bone Marrow is hypoproliferative. While transfusions can improve outcome in the short term, its use increases the risk of infection and mortality without any sustained beneficial effect. The pathogenesis of anemia during sepsis is unclear. High mobility group box 1 (HMGB1), a cytokine that is a critical mediator of sepsis, is released into circulation a few days after sepsis onset, remaining increased for 8 weeks after severe sepsis. HMGB1 levels are increased for at least 8 weeks in murine models of sepsis survival. To induce severe sepsis, cecal ligation and puncture (CLP) was performed in BALB/c mice. Three days after CLP, mice developed persistent anemia, represented by a significant reduction in hematocrit (Sham=49.8±3.2 vs. CLP=29.7±6.7%; p≤0.001), hemoglobin (16.7±1.2 vs. 9.9±2.4mg/dL; p≤0.001), and red blood cells mass (10.2±0.7 vs. 5.4±1.7 x106/µL; p≤0.001). Anemia persisted for at least 25 days after CLP. In CLP survivors, reticulocyte counts were erratic, and insufficient to the degree and duration of anemia (8.2±0.8 vs. 6.6±2.1%; p=ns). Analysis of terminal erythroid differentiation using CD44 and Ter119 or CD44 and FSC as markers demonstrated a significant decrease in all erythroid progenitors, from proerythroblast to orthochromatic erythroblast. Concomitantly, mice surviving CLP developed splenomegaly. Splenic architecture was disrupted after CLP, with expansion of the red pulp, characteristic of stress erythropoiesis. Analysis of terminal erythroid differentiation demonstrated an increase in the quantity of erythroid progenitors. An anti-HMGB1 mAb (2G7) was administered after CLP. Strikingly, 2G7-treated septic mice were significantly protected from developing anemia, and had levels of hemoglobin and hematocrit similar to sham-operated mice. These results highlight a critical role for HMGB1 as key modulator of stress erythropoiesis in a murine model of sepsis survivors. To get further insight into the function of HMGB1 and translate our findings to the pathophysiology of human erythropoiesis, we used CD34+ cells derived from cord blood. Cord blood-derived CD34+ cells were incubated in MethoCult in the presence or not of HMGB1. HMGB1 induced a dose dependent decrease in CFU-E. In murine sepsis, there is a stepwise elevation of different redox forms of HMGB1, with an early increase in all-thiol (inflammatory), followed by a partially oxidized before a fully oxidized (with no known inflammatory activity) appears. At day 7, all-thiol HMGB1 reduced significantly the number of CFU-E, while the fully oxidized had no significant effect. At day 14, the number of BFU-E was reduced in the presence of HMGB1, and further decreased with all-thiol HMGB1. In conclusion, our findings suggest that CLP is a reproducible model to study anemia of sepsis. In mice surviving sepsis, stress erythropoiesis is consistently found. Administration of anti-HMGB1 monoclonal antibody reverses anemia of murine sepsis, demonstrating that HMGB1 can be a potential target in the anemia of sepsis survivors. Translating the findings to the human system, we found that HMGB1 impairs differentiation of CD34+ cells towards the BFU-E and CFU-E stages in colony formation assays, implying that HMGB1 might play a role early during differentiation. The redox status of HMGB1 is critical for its biological function, since its effects are not retrieved when HMGB1 is fully oxidized. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Engraftment kinetics of human CD34+ cells from cord blood and mobilized peripheral blood co-transplanted into NOD/SCID mice

2004 ◽  
Vol 35 (3) ◽  
pp. 271-275 ◽  
Author(s):  
M Ramírez ◽  
C Regidor ◽  
I Marugán ◽  
J García-Conde ◽  
J A Bueren ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Scid Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Kinetics Of ◽  
Mobilized Peripheral Blood

scholarly journals A Comparison of the Kinetics of Nucleated Cells and CD34+ Cells in Neonatal Peripheral Blood and Cord Blood

2007 ◽  
Vol 13 (4) ◽  
pp. 478-485 ◽  
Author(s):  
Joong-Pyo Kim ◽  
Young-Ho Lee ◽  
Young-Ah Lee ◽  
Young-Dae Kim
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Cd34 Cells ◽  
Kinetics Of

scholarly journals Adult peripheral blood and umbilical cord blood NK cells are good sources for effective CAR therapy against CD19 positive leukemic cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
L. Herrera ◽  
S. Santos ◽  
M. A. Vesga ◽  
J. Anguita ◽  
I. Martin-Ruiz ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Peripheral Blood ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Target Cells ◽  
Hematological Cancers ◽  
Adult Peripheral Blood

AbstractAmong hematological cancers, Acute Lymphoblastic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL) are the most common leukemia in children and elderly people respectively. Some patients do not respond to chemotherapy treatments and it is necessary to complement it with immunotherapy-based treatments such as chimeric antigen receptor (CAR) therapy, which is one of the newest and more effective treatments against these cancers and B-cell lymphoma. Although complete remission results are promising, CAR T cell therapy presents still some risks for the patients, including cytokine release syndrome (CRS) and neurotoxicity. We proposed a different immune cell source for CAR therapy that might prevent these side effects while efficiently targeting malignant cells. NK cells from different sources are a promising vehicle for CAR therapy, as they do not cause graft versus host disease (GvHD) in allogenic therapies and they are prompt to attack cancer cells without prior sensitization. We studied the efficacy of NK cells from adult peripheral blood (AB) and umbilical cord blood (CB) against different target cells in order to determine the best source for CAR therapy. AB CAR-NK cells are slightly better at killing CD19 presenting target cells and CB NK cells are easier to stimulate and they have more stable number from donor to donor. We conclude that CAR-NK cells from both sources have their advantages to be an alternative and safer candidate for CAR therapy.

scholarly journals Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes

Blood ◽  
1995 ◽  
Vol 85 (6) ◽  
pp. 1540-1546 ◽  
Author(s):  
C Berthou ◽  
S Legros-Maida ◽  
A Soulie ◽  
A Wargnier ◽  
J Guillet ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Cord Blood ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Cell Subset ◽  
Cytolytic Activity ◽  
Lower Percentage ◽  
Target Cells ◽  
Perforin Expression ◽  
Adult Peripheral Blood

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.

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