Umbilical Cord Blood Hematopoietic Stem/progenitor Cells Research Articles

Induction of AML Preleukemic Fusion Genes in HSPCs and DNA Damage Response in Preleukemic Fusion Gene Positive Samples.

Preleukemic fusion genes (PFGs) occurring after DNA damage in hematopoietic stem progenitor cells (HSPCs) in utero often represent the initial event in the development of childhood leukemia. While the incidence of PFGs characteristic for acute lymphoblastic leukemia (ALL) was relatively well examined by several research groups and estimated to be 1–5% in umbilical cord blood (UCB) of healthy newborns, PFGs that are relevant to acute myeloid leukemia (AML) were poorly investigated. Therefore, this study is focused on the estimation of the incidence of the most frequent AML PFGs in newborns. For the first time, this study considered the inducibility of AML PFGs in different subsets of UCB HSPCs by low-dose γ-rays and also compared endogenous DNA damage, apoptosis, and reactive oxygen species (ROS) level between UCB samples containing or lacking AML PFGs. We found that: (i) the incidence of AML PFGs in UCB was 3.19% for RUNX1-RUNX1T1, 3.19% for PML-RARα, and 1.17% for KMT2A-MLLT3, (ii) 50 cGy of γ-rays did not induce RUNX1-RUNX1T1, PML-RARα, or KMT2A-MLLT3 PFGs in different subsets of sorted and expanded HSPCs, and (iii) the AML PFG+ samples accumulated the same level of endogenous DNA damage, as measured by the γH2AX/53BP1 focus formation, and also the same ROS level, and apoptosis as compared to PFG− controls. Our study provides critical insights into the prevalence of AML PFGs in UCB of newborns, without the evidence of a specific HSPC population more susceptible for PFG formation after irradiation to low-dose γ-rays or increased amount of ROS, apoptosis and DNA damage.

Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal Cells.

In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ease of collection, non-risk and painless harvesting procedure, and lower risk of graft-versus-host disease. However, the therapeutic utility of UCB HSPC has been limited to pediatric patients due to the low cell frequency per unit of UCB. The development of efficient and cost-effective strategies to generate large numbers of functional UCB HSPC ex vivo would boost all current and future medical uses of these cells. Herein, we describe a scalable serum-free co-culture system for the expansion of UCB-derived CD34+-enriched cells using microcarrier-immobilized human bone marrow-derived mesenchymal stromal cells as feeder cells.

Preeclampsia is Associated with Sex-Specific Transcriptional and Proteomic Changes in Fetal Erythroid Cells.

Preeclampsia (PE) has been associated with placental dysfunction, resulting in fetal hypoxia, accelerated erythropoiesis, and increased erythroblast count in the umbilical cord blood (UCB). Although the detailed effects remain unknown, placental dysfunction can also cause inflammation, nutritional, and oxidative stress in the fetus that can affect erythropoiesis. Here, we compared the expression of surface adhesion molecules and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs), UCB erythroid profiles along with the transcriptome and proteome of these cells between male and female fetuses from PE and normotensive pregnancies. While no significant differences were observed in UCB HSPC migration/homing and in vitro erythroid colony differentiation, the UCB HSPC transcriptome and the proteomic profile of the in vitro differentiated erythroid cells differed between PE vs. normotensive samples. Accordingly, despite the absence of significant differences in the UCB erythroid populations in male or female fetuses from PE or normotensive pregnancies, transcriptional changes were observed during erythropoiesis, particularly affecting male fetuses. Pathway analysis suggested deregulation in the mammalian target of rapamycin complex 1/AMP-activated protein kinase (mTORC1/AMPK) signaling pathways controlling cell cycle, differentiation, and protein synthesis. These results associate PE with transcriptional and proteomic changes in fetal HSPCs and erythroid cells that may underlie the higher erythroblast count in the UCB in PE.

Role of RasGRP3 in EPO/EPOR Signaling and Transmigration of Human Hematopoietic CD34+ Cells

Introduction: Umbilical cord blood (UCB) is a salient source of primitive hematopoietic stem progenitor cells (HSPCs) for bone marrow (BM) reconstitution in patients with hematologic and non-hematologic malignancies. However, a relatively low number of HSPCs in UCB units and poor BM homing efficiency greatly hinders the clinical application of UCB CD34+ cells for transplantation. To overcome these hurdles, we developed two independent strategies that increase CD34+ cell numbers and improve BM homing efficiency of UCB HSPCs. First, we expanded UCB HSPCs by culturing them in decellularized Wharton's jelly matrix (DWJM), a biometric scaffold mimicking the 3-dimenstional (3D) microenvironment of BM. Second, we enhanced the in vitro transmigration and in vivo BM homing efficiency of UCB CD34+ cells by blocking EPO/EPOR signaling. Both approaches enhance UCB CD34+ cell migration toward stromal cell-derived factor 1 (SDF1). In this study, we employed RNA-Seq and RT-PCR approaches to analyze UCB HSPCs treated with EPO and co-cultured with DWJM, aiming to identify molecules that regulate UCB HSPC transmigration via EPO/EPOR signaling.Methods: CD34+ cells from highly enriched UCB units (>90% purity) were treated with EPO for 24 hours and separately co-cultured with DWJM for 1 week. UCB CD34+ cells were collected and subjected to RNA-Seq and real-time PCR (RT-PCR) analyses. In vitro transmigration toward SDF-1 was assessed by transwell assay. To assess the involvement of RasGRP3 in UCB CD34+ cell mobility, cells were treated with 100 nM ingenol-3-angelate (I3A), a diacylglycerol (DAG) analog that specifically targets RAS Guanyl Releasing-Protein 3 (RasGRP3), for 16 hours followed by transwell assay. Anti-EPOR antibody-treated or EPO-treated cells were used as controls. In addition, RasGRP3 gene expression was examined in CD34+ cells from peripheral blood (PB) and BM samples collected from the same donor, and compared to RasGRP3 expression in UCB CD34+ cells. Unpaired, 2-tailed t-test was used to analyze results.Results: RasGRP3 was identified by RNA-Seq from the two independent approaches, EPO treatment and DWJM co-culture. EPO downregulated and DWJM upregulated RasGRP3 gene expression in UCB CD34+ cells. RasGRP3 expression was confirmed by qPCR. UCB CD34+ cells that migrated to the bottom chamber of the transwell assays, a population that has a higher mobility, showed an elevated RasGRP3 gene expression and a decreased EPOR cell surface expression. Activation of RasGRP3 by DAG analog I3A induced a significant increase in RasGRP3 gene expression (control: I3A treatment = 1: 202 ± 58, p=0.00012) that was associated with an enhanced transmigration capability (control: I3A = 41%+/-5: 54%+/- 3, p=0.032). Knocking-down of RasGRP3 in K562 cells, a known EPOR expressing cell line, impaired the transmigration capability of K562. CD34+ cells in peripheral blood (PB) showed a higher level of RasGRP3 gene expression compared to CD34+ cells in BM samples from the same healthy donors. RasGRP3 expression in PB CD34+ cells was significantly higher than BM and UCB CD34+ cells (qPCR signals relative to BM, BM: PB: UCB = 1: 431±65: 21±8, p=0.0012, 0.0023, and <0.0001 for BM vs. PB, BM vs. UCB and PB vs. UCB, respectively).Conclusions: By employing transwell assays, flow cytometry and molecular analyses, we demonstrate for the first time that RasGRP3, a protein responsible for GDP/GTP exchange of Ras, regulates the transmigration ability of human CD34+ cells. In addition, our findings connect RasGRP3 expression to the EPOR-mediated signaling pathway in CD34+ cells. A significantly higher level of RasGRP3 expression in PB CD34+ cells than its counterparts in BM might provide an explanation for why PB HSPCs show relatively faster BM engraftment than BM HSPCs during transplantation. Ongoing follow-up studies will elucidate the molecular mechanism(s) underlying EPOR signaling, which holds clinical potential to improve the BM homing deficiency of UCB CD34+ cells via modulating EPOR and RasGRP3 expression (Figure 1). [Display omitted] DisclosuresLiesveld:Onconova: Other: DSMB; Abbvie: Honoraria. Aljitawi:Medpace: Consultancy; The University of Rochester Medical Center: Patents & Royalties: Pending patent related to decellularized Wharton's jelly matrix.

Backtracked analysis of preleukemic fusion genes and DNA repair foci in umbilical cord blood of children with acute leukemia.

The first event in origination of many childhood leukemias is a specific preleukemic fusion gene (PFG) that arises, often in utero, in hematopoietic stem/progenitor cells (HSPC) from misrepaired DNA double strand break (DSB). An immanently elevated level of DSB and impaired apoptosis may contribute to origination and persistence of PFG and donor cell-derived leukemia in recipients of allogeneic transplantation of umbilical cord blood (UCB). We investigated DSB, apoptosis and PFG in the backtracked UCB cells of leukemic patients. RNA from UCB of three patients with acute lymphoblastic leukemia, patient with acute megakaryoblastic leukemia and Down syndrome, and four healthy children was screened for common PFG by RT-qPCR. Presence of PFG was validated by sequencing. Endogenous γH2AX and 53BP1 DNA repair foci, cell populations, and apoptosis were analyzed in UCB CD34+/- cells with imaging and standard flow cytometry. We found MLL2-AF4 and BCR-ABL (p190) fusion genes in UCB of two out from four pediatric patients, apparently not detected at diagnosis, while UCB cells of TEL-AML1+ ALL patient were tested negative for this PFG and no PFG were detected in UCB cells of healthy children. No significant difference in DNA damage and apoptosis between UCB CD34+/- cells from healthy children and leukemic patients was observed, while Down syndrome trisomy increased DNA damage and resulted in distribution of cell populations resembling transient abnormal myelopoiesis. Our findings indicate increased genetic instability in UCB HSPC of leukemic patients and may be potentially used for diagnostics and exclusion of possibly affected UCB from transplantation.

Initial CD34+ cell‐enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon Ex vivo expansion

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal-based serum-free culture system to evaluate the effect of different initial CD34(+) cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34(+) and CD34(+) CD90(+) expression, we have identified early activation of CD34 expression on CD34(-) cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34(+) cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34(+)/CD34(+) CD90(+) cell yield (High: 14 ± 1.0/3.5 ± 1.4-fold; Medium: 22 ± 2.0/3.4 ± 1,0-fold; Low: 31 ± 3.0/4.4 ± 1.5-fold) after a 7-day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34(+) cell recovery for each strategy, on overall CD34(+) cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34(+) cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10(6) cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex-vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost-effective expansion of HSC for cellular therapy.

Microencapsulated Osteoblasts Support Hematopoietic Stem/Progenitor Cell Expansion in Hypoxic Environment*

Microencapsulated osteoblasts were cocultured with hematopoietic stem/progenitor cells (HSPCs) under hematopoietic niche oxygen concentration to investigate the promoting effort of hematopoietic microenvironment on the expansion of umbilical cord blood HSPCs. The osteobalsts were isolated from human iliac bone and cultured, the third passage of osteoblasts at a density of 8 ×105 cells/ml were encapsulated in gelatin-alginiate-chitosan (GAC) beads with a diameter of 0.5 mm by the polyelectrolyte-complexation method. Three groups of cells were cultured in 5% oxygen incubator, A′ group with microencapsulated osteoblasts and hematopoietic cells, B′ with only hematopoietic cells and C′ with only microencapsulated osteoblasts. Meanwhile, the similarly grouped cells were cultured under 20% oxygen condition, named as A, B and C groups, respectively. The expansion of HSPCs was evaluated by flow cytometry analysis and colony-forming assays. And the concentrations of two kinds of cytokines, LIF and IL-6, were tested to investigate the mechanism of osteoblast's action. The results showed that human osteoblasts dispersed uniformly and grew well in microbeads. There were amount of micro holes in the beads for nutrients transmission. Lots of hematopoietic cells adhered weakly on the surface of microbeads. After 7 days of culture, the hematopoietic cell expansion folds were (49.0 ± 4.6), (3.3 ± 0.5), (17.7 ± 1.2) and (1.9 ± 0.2) respectively for group A′, B′, A and B. And CD34+ cells in groups A′, B′ and A were expanded (87.6 ± 8.3)-fold, (2.2 ± 0.3)-fold and (14.9 ± 1.0)-fold, but CD34+ cells in group B descended. CFU-Cs expansion folds in group A′, B′, A and B were (9.8 ± 0.8), (3.5 ± 0.4), (6.9 ± 0.7) and (2.6 ± 0.2) respectively. It was indicated that Hypoxic co-culture system could promote HSPCs expansion much more than normoxic co-culture system and somatic cell-free culture system. IL-6 and LIF concentrations in A′ , B′ and C′ were significantly higher than those in groups A, B and C, which were consistent with their expansion results. Moreover, microencapsulated osteoblasts could support the expansion of umbilical cord blood HSPCs, especially in 5% oxygen condition. Osteoblasts lived in low oxygen condition could secrete more cytokines and thus regulate HSPCs expansion.

Novel Mechanistic Insight Into Mobilization of Hematopoietic Stem/Progenitor Cells (HSPCs): Complement Cascade and Membrane Attack Complex Activated in Bone Marrow Sinusoids During Mobilization Release From Erythrocytes Sphingosine-1 Phosphate – An Underappreciated Chemoattractant Executing Egress of HSPCs.

Abstract 31We reported that complement cascade (CC) is activated in bone marrow (BM) during mobilization of hematopoietic stem/progenitor cells (HSPCs) and that CC clevage fragments direct egress of HSPCs from BM into peripheral blood (PB) (Blood 2003;101,3784; Blood 2004;103,2071; Blood 2005;105,40). We also reported that C5 cleavage fragments play a crucial role in the mobilization process by: i) inducing proteolytic activity in the BM environment; ii) directing BM egress of granulocytes that “pave a road” for HSPCs; and iii) inducing secretion of cationic peptides from activated granulocytes that prime HSPC egress (Leukemia 2009; in press). In this study, we sought to determine which major chemottractant is present in PB that is responsible for egress of HSPCs and whether activation of CC plays some role in its level/expression. We noticed that plasma derived from normal and mobilized PB strongly chemoattracts murine and human HSPCs. This chemotactic effect was not dependent on plasma SDF-1 levels because: i) it occurs unaffectedly in the presence of CXCR4 antagonist AMD3100; ii) it was still robust to heat-inactivated sera; and iii) ELISA studies revealed negligible concentrations of SDF-1, which did not correlate with good or poor mobilizer status. However, to our surprise, we noticed that plasma isolated from G-CSF-mobilized mice and patients contains traces of free hemoglobin, which suggests some level of hemolysis occurs in mobilized PB. As such, we performed chemotactic assays in the presence of different concentrations of lysed erythrocytes and noticed that such diluted lysates are potent chemoattractants for HSPCs. The chemotactic activities of these lysates were resistant to heat inactivation similarly as patient sera. Based on this, we focused on S1P, a thermo-resistant lipid that, as reported, chemoattracts HSPCs and whose major reservoirs are erythrocytes (FASEB J 2007:21;1202). In fact we found by ELISA that S1P level increases during mobilization in PB and that SP1 is the most potent chemoattractant for BM-residing HSPCs, much stronger than SDF-1 - if both compounds are compared in physiologically relevant concentrations. Furthermore, activation of S1P receptors on BM-derived HSPCs augmented responsiveness to SDF-1 gradient up to 50%. However, these chemotactic effects of S1P were not visible for previously mobilized PB or umbilical cord blood HSPCs, which we explain by a fact that these mobilized cells are already desensitized to S1P gradient. Therefore, we propose the following scenario. First, a mobilizing agent (e.g., G-CSF) induces activation of CC in BM that subsequently contributes to the release of protelolytic enzymes from granulocytes that perturb SDF-1-CXCR4/VLA-4-VCAM1 interactions and stimulate egress of activated granulocytes from BM that “pave a road” for egress of HSPCs. Simultaneously, the final product of CC activation (C5b-C9), the membrane attack complex (MAC), induces in BM sinusoids the release of S1P from erythrocytes. S1P accumulating in BM sinusoids and cationic peptides released from activated granulocytes, but not changes in plasma SDF-1 levels, are crucial executors of HSPCs egress from BM into PB. Thus, our results provide novel evidence that CC activation/membrane attack complex (MAC)-induced elevated plasma S1P level is essential for egress/mobilization of HSPCs. Disclosures:No relevant conflicts of interest to declare.

Biology of human umbilical cord blood-derived hematopoietic stem/progenitor cells.

Reported in 1989, studies by Broxmeyer, Gluckman, and colleagues demonstrated that umbilical cord blood (UCB) is a rich source of hematopoietic stem/progenitor cells (HSPC) and that UCB could be used in clinical settings for hematopoietic cell transplantation. Since then, a great interest has been generated on the biological characterization of these cells. Over the last nine years, several groups have focused on the study of UCB HSPC, addressing different aspects, such as the frequency of these cells in UCB, the identification of different HSPC subsets based on their immunophenotype, their ability to respond to hematopoietic cytokines, the factors that control their proliferation and expansion potentials, and their capacity to reconstitute hematopoiesis in animal models. Most of these studies have shown that significant functional differences exist between HSPC from UCB and adult bone marrow (i.e., the former possess higher proliferation and expansion potential than the latter). It is also noteworthy that genetic manipulation of UCB HSPC has been achieved by several groups and that genetically modified UCB cells have already been used in the clinic. In spite of the significant advances in the characterization of these cells, we are still in the process of trying to fully understand their biology, both at the cellular and the molecular levels. In the present article, we describe and discuss what is currently known about the biology of UCB HSPC.