Cord Blood CD34 Research Articles

Abstract 3 Generation of Myeloid Cells from Cord Blood-Derived CD34+ Cells for Therapeutic Intent

IntroductionMacrophages and monocytes traffic to and infiltrate complex solid tumor microenvironments where conditions can promote an immunosuppressive phenotype in resident tumor-associated macrophages that hinder effective anti-tumor immune responses. Ongoing advances in cell engineering are being adapted to modify monocytes and macrophages into efficacious anti-tumor cell therapies. However, a limiting factor to this promising therapy has been cell numbers, since monocytes obtained from adult peripheral blood mononuclear cells do not expand ex vivo.ObjectiveWe are developing an expanded allogeneic, cryopreserved, off-the-shelf myeloid cell product for the treatment of solid tumors.MethodsCD34+ cells isolated from pooled donor cord blood are the starting material for generation of myeloid cells. The process has three phases: (1) expansion of hematopoietic stem and progenitor cells, (2) continued expansion and differentiation into monocytes, and (3) differentiation of monocytes into mature macrophages. Cells are characterized before and after cryopreservation by immune phenotyping, morphology, and phagocytic capacity.ResultsThe current culture method produces several thousand HLA-DR+ CD11b+ myeloid cells at both the monocyte and macrophage stage per starting CD34+ cell. These cells make up ~70% of the final cell product along with a potentially beneficial heterogeneous mix of additional myeloid-derived cells. Immunophenotyping demonstrates expression of canonical monocyte/macrophage markers, including CD14, CD86, and CD163. Cell morphology after macrophage differentiation is similar to that of peripheral blood-derived mature adult macrophages. Importantly, the final cell product can be cryopreserved with excellent recovery of viable cells post-thaw that are functional, as demonstrated by maintenance of phagocytic potential when compared with pre-cryopreservation function.DiscussionThis proprietary CD34+ cell expansion and directed differentiation platform results in the generation of therapeutically relevant numbers of functional myeloid cells. Furthermore, the ability to cryopreserve these cells, with demonstrated viability and function upon thaw, facilitates their use as an off-the-shelf cell therapy, with potential for faster and less expensive routes to treatments for solid tumors that have proven resistant to other cell therapies. Additional work is being done toward engineering these myeloid cells to express proteins to enhance a patient’s anti-tumor immune responses, whether these cells are used alone or in combination with other therapeutics.

Abstract 22 CellStor Transfer Freezing Bag: Reliable Closed System for Complex Cord Blood Sample Processing and CD34+ Hematopoietic Stem Cell Cryopreservation

IntroductionHematopoietic stem cell (HSC) transplants are effective in the treatment of numerous disorders. Proper processing and storage of umbilical cord blood (UCB) and peripheral blood (PB) HSCs is invaluable for therapeutic applications. Although automated sample processing methods exist, they are unable to accommodate low volume, viscous, fibrin clotted, partially hemolyzed, or otherwise complex samples, resulting in the loss of precious HSCs. The CellBios single-use transfer freezing bag was designed to process peripheral and cord blood samples and freeze HSCs, including from those with the above-mentioned problems. Here we demonstrate the utility of these transfer freezing bags to salvage problematic UCB samples and recover precious HSC material suitable for banking and therapeutics.ObjectiveThe objective of this study was to develop a closed UCB sample processing and cryopreservation solution that is amenable to easy manual operation, can deliver excellent HSC viability and recovery, can eliminate contaminating red blood cells (RBCs), and is capable of processing complex/defective starting samples that are incompatible with conventional automated processing systems into therapeutic grade HSC banks.MethodsWe studied 100 randomly selected UCB samples, from various centers throughout India, that were processed and prepared for cryopreservation using our transfer freezing bag. We recorded the pre- and post-sample processing volumes, total nucleated cell counts (TNCC), hematocrit (HCT), CD34+ cell viability, and recovery.ResultsStudy results showed the following: 94% vs 98% CD34+ cell viability post processing, 77% vs 85% total mononuclear cell viability, 69% vs 85% TNCC recovery in problematic vs normal samples, respectively. It was possible to achieve low RBC carryover –91% HCT reduction post-processing in normal samples; more importantly, problematic samples also had RBC removal evidenced by HCT reduction of 45%.DiscussionWe have demonstrated efficient removal of RBCs to alleviate post-transplantation issues and maximized CD34+ HSC recovery in both normal and problematic UCB samples. Crucially, it was possible to salvage precious CB HSCs from defective samples that would be lost had they been processed in conventional automated workflows. The CellBios CellStor Transfer Freezing Bag Set offers best in class performance in preparing umbilical cord blood samples for cryostorage.

ANTXR1 Regulates Erythroid Cell Proliferation and Differentiation through wnt/β-Catenin Signaling Pathway In Vitro and in Hematopoietic Stem Cell.

Erythropoiesis is a highly complex and sophisticated multistage process regulated by many transcription factors, as well as noncoding RNAs. Anthrax toxin receptor 1 (ANTXR1) is a type I transmembrane protein that binds the anthrax toxin ligands and mediates the entry of its toxic part into cells. It also functions as a receptor for the Protective antigen (PA) of anthrax toxin, and mediates the entry of Edema factor (EF) and Lethal factor (LF) into the cytoplasm of target cells and exerts their toxicity. Previous research has shown that ANTXR1 inhibits the expression of γ-globin during the differentiation of erythroid cells. However, the effect on erythropoiesis from a cellular perspective has not been fully determined. This study examined the role of ANTXR1 on erythropoiesis using K562 and HUDEP-2 cell lines as well as cord blood CD34+ cells. Our study has shown that overexpression of ANTXR1 can positively regulate erythrocyte proliferation, as well as inhibit GATA1 and ALAS2 expression, differentiation, and apoptosis in K562 cells and hematopoietic stem cells. ANTXR1 knockdown inhibited proliferation, promoted GATA1 and ALAS2 expression, accelerated erythrocyte differentiation and apoptosis, and promoted erythrocyte maturation. Our study also showed that ANTXR1 may regulate the proliferation and differentiation of hematopoietic cells, though the Wnt/β-catenin pathway, which may help to establish a possible therapeutic target for the treatment of blood disorders.

Remodeled CD146+CD271+ Bone Marrow Mesenchymal Stem Cells from Patients with Polycythemia Vera Exhibit Altered Hematopoietic Supportive Activity.

An essential component of the hematopoietic microenvironment, bone marrow mesenchymal stem cells (BM-MSCs) play an important role in the homeostasis and pathogenesis of the hematopoietic system by regulating the fate of hematopoietic stem cells (HSCs). Previous studies revealed that BM-MSCs were functionally remodeled by malignant cells in leukemia. However, the alterations in BM-MSCs in polycythemia vera (PV) and their effects on HSCs still need to be elucidated. Our results demonstrated that although BM-MSCs from PV patients shared similar surface markers with those from healthy donors, they exhibited enhanced proliferation, decreased senescence, and abnormal osteogenic differentiation capacities. The CD146+CD271+ BM-MSC subpopulation, which is considered to give rise to typical cultured BM-MSCs and form bone and the hematopoietic stroma, was then sorted. Compared with those from healthy donors, CD146+CD271+ BM-MSCs from PV patients showed an impaired mesensphere formation capacity and abnormal differentiation toward osteogenic lineages. In addition, CD146+CD271+ PV BM-MSCs showed altered hematopoietic supportive activity when cocultured with cord blood CD34+ cells. Our study suggested that remodeled CD146+CD271+ BM-MSCs might contribute to the pathogenesis of PV, a finding that will shed light on potential therapeutic strategies for PV.

Higher Cryopreserved CD34+ Cell Dose Is Associated with Decreased Hepatic Veno-Occlusive Disease/Sinusoidal Obstruction Syndrome after Single-Unit Cord Blood Transplantation in Adults Given Prophylactic Ursodeoxycholic Acid and Intravenous Heparin

Hepatic veno-occlusive disease (VOD), also known as sinusoidal obstruction syndrome (SOS), is one of the most serious complications to occur early after allogeneic hematopoietic cell transplantation (HCT). However, detailed data on VOD/SOS after cord blood transplantation (CBT) are not available. The objective of this retrospective study was to evaluate the incidence, risk factors, and clinical impact of VOD/SOS after single-unit unrelated CBT for adult patients at our institution. We retrospectively evaluated the incidence, risk factors, and outcomes of VOD/SOS after 390 single-unit unrelated CBTs performed in 332 adults under a prophylactic strategy of ursodeoxycholic acid (UDCA) and i.v. heparin at our institution between 1998 and 2021. VOD/SOS was observed in 24 of the 390 CBTs. The cumulative incidence of VOD/SOS was 5.9% at 30 days and 6.2% at 100 days after CBT. Multivariate analysis showed that cryopreserved CD34+ cell dose ≥1.0×105/kg was significantly associated with a decreased risk of VOD/SOS after CBT (hazard ratio [HR], 0.33; 95% confidence interval [CI], 0.12 to 0.91; P=.032). In multivariate analysis, the development of VOD/SOS was significantly associated with higher overall mortality (HR, 6.19; 95% CI, 3.61 to 10.65; P < .001), treatment failure (HR, 4.79; 95% CI, 2.95 to 7.76; P < .001), and nonrelapse mortality (HR, 12.60; 95% CI, 6.90 to 23.00; P < .001). Our study shows that the incidence of hepatic VOD/SOS was relatively low after unrelated single-unit CBT under a prophylactic strategy of UDCA and i.v. heparin. A higher cryopreserved cord blood CD34+ cell dose was associated with a reduction in VOD/SOS, suggesting that selection of a higher cord blood unit CD34+ cell dose could be efficient in preventing hepatic VOD/SOS in adults undergoing single-unit CBT.

Transmembrane Protein ANTXR1 Regulates γ-Globin Expression by Targeting the Wnt/β-Catenin Signaling Pathway.

Reactivation of fetal hemoglobin (HbF, α2γ2) alleviates clinical symptoms in patients with β-thalassemia and sickle cell disease, although the regulatory mechanisms of γ-globin expression have not yet been fully elucidated. Recent studies found that interfering with the expression of the membrane protein ANTXR1 gene upregulated γ-globin levels. However, the exact mechanism by which ANTXR1 regulates γ-globin levels remains unclear. Our study showed that overexpression and knockdown of ANTXR1 in K562, cord blood CD34+, and HUDEP-2 cells decreased and increased γ-globin expression, respectively. ANTXR1 regulates the reactivation of fetal hemoglobin (HbF, α2γ2) in K562, cord blood CD34+, and adult peripheral blood CD34+ cells through interaction with LRP6 to promote the nuclear entry of β-catenin and activate the Wnt/β-catenin signaling pathway. The overexpression or knockdown of ANTXR1 on γ-globin and Wnt/β-catenin signaling in K562 cells was reversed by the inhibitor XAV939 and the activator LiCl, respectively, where XAV939 inhibits the transcription of β-catenin in the Wnt pathway, but LiCl inhibits GSK3-β. We also showed that the binding ability of the rank4 site in the transcriptional regulatory region of the SOX6 gene to c-Jun was significantly increased after overexpression of ANTXR1 in K562 cells. SOX6 protein expression was increased significantly after overexpression of the c-Jun gene, indicating that the transcription factor c-Jun initiated the transcription of SOX6, thereby silencing γ-globin. Our findings may provide a new intervention target for the treatment of β-hemoglobinopathies.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A

BackgroundHuman erythropoiesis is a tightly regulated, multistep process encompassing the differentiation of hematopoietic stem cells (HSCs) toward mature erythrocytes. Cellular metabolism is an important regulator of cell fate determination during the differentiation of HSCs. However, how O-GlcNAcylation, a posttranslational modification of proteins that is an ideal metabolic sensor, contributes to the commitment of HSCs to the erythroid lineage and to the terminal erythroid differentiation has not been addressed.MethodsCellular O-GlcNAcylation was manipulated using small molecule inhibition or CRISPR/Cas9 manipulation of catalyzing enzyme O-GlcNAc transferase (OGT) and removing enzyme O-GlcNAcase (OGA) in two cell models of erythroid differentiation, starting from: (i) human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) to investigate the erythroid lineage specification and differentiation; and (ii) human-derived erythroblastic leukemia K562 cells to investigate the terminal differentiation. The functional and regulatory roles of O-GlcNAcylation in erythroid differentiation, maturation, and globin production were investigated, and downstream signaling was delineated.ResultsFirst, we observed that two-step inhibition of OGT and OGA, which were established from the observed dynamics of O-GlcNAc level along the course of differentiation, promotes HSPCs toward erythroid differentiation and enucleation, in agreement with an upregulation of a multitude of erythroid-associated genes. Further studies in the efficient K562 model of erythroid differentiation confirmed that OGA inhibition and subsequent hyper-O-GlcNAcylation enhance terminal erythroid differentiation and affect globin production. Mechanistically, we found that BCL11A is a key mediator of O-GlcNAc-driven erythroid differentiation and β- and α-globin production herein. Additionally, analysis of biochemical contents using synchrotron-based Fourier transform infrared (FTIR) spectroscopy showed unique metabolic fingerprints upon OGA inhibition during erythroid differentiation, supporting that metabolic reprogramming plays a part in this process.ConclusionsThe evidence presented here demonstrated the novel regulatory role of O-GlcNAc/BCL11A axis in erythroid differentiation, maturation, and globin production that could be important in understanding erythropoiesis and hematologic disorders whose etiology is related to impaired erythroid differentiation and hemoglobinopathies. Our findings may lay the groundwork for future clinical applications toward an ex vivo production of functional human reticulocytes for transfusion from renewable cell sources, i.e., HSPCs and pluripotent stem cells.

Maternal High-Fat Diet Aggravates Allergic Asthma in Offspring via Modulating CD4+ T-Cell Differentiation.

Maternal improper nutrition has been reported to trigger respiratory disorders in offspring. Here, we characterized the effects of high-fat environment in the fetal period on mice and human cord blood CD4+ T-lymphocytes, and investigated their roles in susceptibility to asthma. Mice born to mothers that consumed a high-fat diet (HFD) throughout the gestation period were sensitized by ovalbumin to establish an experimental asthma model. To further extrapolate to humans, we collected cord blood from neonates of hypercholesterolemic (HC) mothers (n = 18) and control mothers (n = 20). In mice, aggravated airway hyperresponsiveness and inflammation revealed that maternal high-fat diet could lead to exacerbated allergic asthma in adult offspring. It was partially due to augmented activation and proliferation of CD4+ T-cells, where upregulated klf2 mRNA levels may be potentially involved. Notably, naïve HFD CD4+ T-cells had enhanced TH2-based immune response both in vivo and in vitro, resulting from DNA hypomethylation of the Il-4 promoter region. Moreover, in human, TH2 cytokines transcripts were enhanced in CD4+ T-cells of the HC group, which was associated with an increased risk of developing allergic diseases at 3 years old. Together, our study indicated that early life improper nutrition-triggered epigenetic changes in T-cells may contribute to long-lasting alterations in allergic diseases.

Abstract 1640: Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L

Abstract Humanized mice are being applied widely to study human immune system homeostasis, function, and as a testing platform for cancer immunotherapies. A major limitation for many humanized mouse models is the lack of functional and mature human innate immune cells, which are critical for effective human immune system-tumor interactions. Previous studies have demonstrated that delivery of human FLT3L into immunodeficient mice that lack mouse FLT3, promotes the development of human innate immune cell subsets following engraftment with human hematopoietic stem cells (HSC). Here we describe a NOD-scid IL2rgnull (NSG) mouse that lacks the expression of mouse FLT3 and expresses human FLT3L transgenically (NSG-mFLT3nullTg (HuFLT3L) or NSG-FLT3L. In these studies, NSG-FLT3L and NSG mice were engrafted with human umbilical cord blood (UCB) CD34+ HSC and compared for human immune system development and function. HSC-engrafted NSG-FLT3L and NSG mice show similar levels of total human CD45+ cells in blood over the course of 18 weeks post-engraftment. However, HSC-engrafted NSG-FLT3L mice show significantly higher levels of human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes, CD56+ NK cells and CD3+ T cells in the blood as compared to NSG mice. CD34-FLT3L mice also show increased levels of human immune cell infiltration into the gut mucosa. CD56+ NK cells and CD3+ T cells from HSC-engrafted NSG-FLT3L mice express granzyme A and granzyme B, indicating cytotoxic activity. Following treatment of HSC-engrafted NSG-FLT3L mice with LPS, heightened levels of human cytokines were detected in serum samples, confirming innate immune system function. The growth kinetics of tumor cells from a triple negative breast cancer cell line MDA-MB-231 and a lung PDX model LG1306 in HSC-engrafted NSG-FLT3L mice are work in progress and will be compared with HSG-engrafted NSG mice. Overall these results demonstrate that the NSG-FLT3L model supports enhanced development of functional, human innate immune cells and is a novel tool to study human immuno-oncology. Citation Format: Li-Chin Yao, Shantashri Vaidya, Pali Kaur, James G. Keck, Leonard D. Shultz, Dale L. Greiner, MIchael A. Brehm. Enhanced development of functional human innate immune cells in a novel FLT3nullNSG mouse strain expressing human FLT3L [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1640.

Abstract 1639: Human immune system mouse model of ER+ metastatic breast cancer

Abstract In a recent clinical trial in heavily pretreated estrogen receptor alpha-positive (ER+) metastatic breast cancer (MBC), ~23% of patients reached the primary endpoint of clinical benefit of &amp;gt;24 weeks on Fulvestrant plus the anti-androgen Enzalutamide. ESR1 mutant biopsies had more T regulatory cells (t test p&amp;lt;0.021) and macrophages (p&amp;lt;0.003) than ESR1 wild type (WT), which had more TP53 mutations and higher levels of cytotoxic T effector cells (p&amp;lt;0.01). In addition, PD-L1 increased in post- vs. pre-treatment biopsies (paired t test p&amp;lt;0.002). This increase in PD-L1 post-treatment warrants testing if the addition of a checkpoint inhibitor to the Fulvestrant-Enzalutamide combination will enhance therapy efficacy in ER+ MBC resistant to standard endocrine therapy. However, the lack of immune competent ER+ breast cancer pre-clinical models is a major barrier to these studies. Human immune system (HIS) mouse models were developed as a platform in which to test immune checkpoint inhibitors in other human cancers, and one HIS model was recently shown to support the growth and immune infiltration of ER+ patient-derived xenograft (PDX) primary tumors. We tested whether HIS mice are a robust model in which to define ER+ tumor-immune microenvironment interactions at metastatic sites. We implanted NCG mice (NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl) humanized by engraftment of human umbilical cord blood-derived CD34+ (huCD34+) stem cells with the estrogen-independent HCI-013EI (013EI) PDX, derived from an ER+, invasive lobular MBC which harbors the Y537S ESR1 mutation. Mice were monitored for tumor latency and growth rate, and primary tumors and organs were collected for immunohistochemistry, single-cell RNA sequencing (scRNAseq), and phasor-fluorescence lifetime imaging microscopy (FLIM). The ER mutated PDX 013EI had a longer tumor latency in HIS (median 77 days) vs. age-matched immune deficient control mice (27.5 days, log-rank p=0.043). Once formed, 013EI tumors in HIS mice grew slower than those in controls (mixed-effects analysis, p=0.035). HIS mice had visible lung, liver, and/or gastrointestinal tract metastases despite small primary tumors. Lung metastases from HIS mice retain ER expression and are infiltrated by human CD4+, CD8+, and CD68+ immune cells. Initial scRNAseq analysis also suggests infiltration by diverse immune cell populations in primary tumors from HIS mice. Initial phasor-FLIM for NADH autofluorescence suggests that 013EI tumors are highly glycolytic in HIS mice. In conclusion, our results show that huCD34+ HIS mice effectively support growth, metastasis, and immune infiltration of an ESR1 mutant MBC PDX. Ongoing studies will further define the tumor-immune microenvironment in this and ESR1 WT MBC PDX, focusing on immune cell infiltration, PD-L1 expression, steroid receptor status, metabolic signaling and changes in these parameters following novel combinations of endocrine, immunotherapy and metabolic agents. Citation Format: Ayodeji Olukoya, Shaymaa Bahnassy, Nicole S. Spoelstra, Lu Jin, Suman Ranjit, Anthony Elias, Jennifer K. Richer, Rebecca B. Riggins. Human immune system mouse model of ER+ metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1639.

The upregulation of Gata transcription factors family and FOG-1 in expanded and differentiated cord blood-derived CD34+ hematopoietic stem cells to megakaryocyte lineage during co-culture with cord blood mesenchymal stem cells

BackgroundUmbilical cord blood (UCB) has improved into an attractive and alternative source of allogeneic hematopoietic stem cells (all-HSCs) in clinics and, research for three decades. Recently, it has been shown that the limited cell dose of, this valuable source can be enhanced by the ex vivo expansion of cells in many, ways. We evaluated the expression of the Gata transcription factors family and FOG-1, in expanded and differentiated cord blood-derived CD34 + hematopoietic stem cells to, megakaryocytes lineage., Methods: Separated mononuclear cells were cultured in DMEM complete medium., Harvested cells as a mesenchymal stem cell at 85 % confluency were cultured with, trypsin/EDTA and in 24-well plates. The characteristic analyses of isolated UCB- MSCs, were done by flow cytometry and adipogenic, chondrogenic, and osteogenic, differentiation assays. MACS purified UCB-CD34 + hematopoietic cells cultivated and, differentiated to megakaryocyte progenitor cells in the presence of cytokine cocktail, with UCB-MSCs. Then, the GATA1, GATA2, GATA3, and FOG-1 genes expression, after differentiation to megakaryocyte progenitor cells were performed by quantitative, real-time polymerase chain reaction (PCR)., Results: In this study, the results of real-time-PCR showed that the fold change, expression of GATA-1, FOG-1, and GATA-2 genes after co-culturing with UCB-MSCs, significantly increased to 7.3, 4.7, and 3.3-fold in comparison with control groups;respectively., Conclusion: UCB-MSCs can increase the expansion and differentiation of UCBCD34 + , to megakaryocyte progenitor cells through upregulation of GATA-1, GATA-2, and FOG-1 gene expression.

RUNX1 Inhibition Using Lipid Nanoparticle-Mediated Silencing RNA Delivery as an Effective Treatment for Acute Leukemias

Transcription factor RUNX1 plays key roles in the establishment and maintenance of the hematopoietic system. Although RUNX1 has been considered a beneficial tumor suppressor, several recent reports have described the tumor-promoting role of RUNX1 in a variety of hematopoietic neoplasms. In this study, we assessed the effect of RUNX1 depletion in multiple human leukemia cell lines using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system, and confirmed that RUNX1 is in fact required for sustaining their leukemic proliferation. To achieve efficient RUNX1 inhibition in leukemia cells, we then examined the effect of lipid nanoparticle (LNP)-mediated delivery of RUNX1-targeting small interfering (si)RNA using two tumor-tropic LNPs. The LNPs containing RUNX1-targeting siRNA were efficiently incorporated into myeloid and T-cell leukemia cell lines and patient-derived primary human acute myeloid leukemia (AML) cells, downregulated RUNX1 expression, induced cell cycle arrest and apoptosis, and exhibited the growth-inhibitory effect in them. In contrast, the LNPs were not efficiently incorporated into normal cord blood CD34+ cells, indicating their minimum cytotoxicity. Thus, our study highlights RUNX1 as a potential therapeutic target to inhibit leukemogenesis, and provides the LNP-based siRNA delivery as a promising approach to deplete RUNX1 specifically in leukemia cells.

Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment.

The widespread clinical application of cord blood (CB) for hematopoietic stem cell (HSC) transplantation is limited mainly by the inadequate number of hematopoietic stem and progenitor cells (HSPCs) in single CB units, which results in unsuccessful or delayed engraftment in recipients. The identification of agents to promote CB HSPC engraftment has significant therapeutic value. Here, we found that transient inhibition of the JNK pathway increased the HSC frequency in CB CD34+ cells to 13.46-fold. Mechanistic studies showed that inhibition of the JNK pathway upregulated the expression of quiescence-associated and stemness genes in HSCs, preventing HSCs from entering the cell cycle, increasing glucose uptake and accumulating reactive oxygen species (ROS). Importantly, transient inhibition of the JNK pathway during CB CD34+ cell collection also enhanced long-term HSC (LT-HSC) recovery and engraftment efficiency. Collectively, these findings suggest that transient inhibition of the JNK pathway could promote a quiescent state in HSCs by preventing cell cycle entry and metabolic activation, thus enhancing the HSC number and engraftment potential. Together, these findings improve the understanding of the regulatory mechanisms governing HSC quiescence and stemness and have the potential to improve HSC collection and transplantation.

Regulatory Crosstalk between Physiological Low O2 Concentration and Notch Pathway in Early Erythropoiesis.

Physiological low oxygen (O2) concentration (<5%) favors erythroid development ex vivo. It is known that low O2 concentration, via the stabilization of hypoxia-induced transcription factors (HIFs), intervenes with Notch signaling in the control of cell fate. In addition, Notch activation is implicated in the regulation of erythroid differentiation. We test here if the favorable effects of a physiological O2 concentration (3%) on the amplification of erythroid progenitors implies a cooperation between HIFs and the Notch pathway. To this end, we utilized a model of early erythropoiesis ex vivo generated from cord blood CD34+ cells transduced with shHIF1α and shHIF2α at 3% O2 and 20% O2 in the presence or absence of the Notch pathway inhibitor. We observed that Notch signalization was activated by Notch2R–Jagged1 ligand interaction among progenitors. The inhibition of the Notch pathway provoked a modest reduction in erythroid cell expansion and promoted erythroid differentiation. ShHIF1α and particularly shHIF2α strongly impaired erythroid progenitors’ amplification and differentiation. Additionally, HIF/NOTCH signaling intersects at the level of multipotent progenitor erythroid commitment and amplification of BFU-E. In that, both HIFs contribute to the expression of Notch2R and Notch target gene HES1. Our study shows that HIF, particularly HIF2, has a determining role in the early erythroid development program, which includes Notch signaling.

Analyses of erythropoiesis from embryonic stem cell-CD34+ and cord blood-CD34+ cells reveal mechanisms for defective expansion and enucleation of embryomic stem cell-erythroid cells.

Red blood cells (RBCs) generated ex vivo have the potential to be used for transfusion. Human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) possess unlimited self‐renewal capacity and are the preferred cell sources to be used for ex vivo RBC generation. However, their applications are hindered by the facts that the expansion of ES/iPS‐derived erythroid cells is limited and the enucleation of ES/iPS‐derived erythroblasts is low compared to that derived from cord blood (CB) or peripheral blood (PB). To address this, we sought to investigate the underlying mechanisms by comparing the in vitro erythropoiesis profiles of CB CD34+ and ES CD34+ cells. We found that the limited expansion of ES CD34+ cell‐derived erythroid cells was associated with defective cell cycle of erythroid progenitors. In exploring the cellular and molecular mechanisms for the impaired enucleation of ES CD34+ cell‐derived orthochromatic erythroblasts (ES‐ortho), we found the chromatin of ES‐ortho was less condensed than that of CB CD34+ cell‐derived orthochromatic erythroblasts (CB‐ortho). At the molecular level, both RNA‐seq and ATAC‐seq analyses revealed that pathways involved in chromatin modification were down‐regulated in ES‐ortho. Additionally, the expression levels of molecules known to play important role in chromatin condensation or/and enucleation were significantly lower in ES‐ortho compared to that in CB‐ortho. Together, our findings have uncovered mechanisms for the limited expansion and impaired enucleation of ES CD34+ cell‐derived erythroid cells and may help to improve ex vivo RBC production from stem cells.

Human CD4 T Cells From Thymus and Cord Blood Are Convertible Into CD8 T Cells by IL-4.

Commitment to the CD4+ or CD8+ T cell lineages is linked to the acquisition of a functional program broadly defined by helper and cytotoxic properties, respectively. The mechanisms underlying these processes in the human thymus remain largely unclear. Moreover, recent thymic emigrants are thought to have some degree of plasticity, which may be important for the shaping of the immune system and adjustment to specific peripheral needs. We show here that IL-4 induces proliferation-independent de novo synthesis of CD8αβ in human CD4 single-positive (SP) thymocytes, generating a stable CD8SP population that features a diverse TCRαβ repertoire, CD4 expression shut-down and ThPOK downregulation. IL-4 also promotes an innate-like program in both CD4SP and CD8SP thymocytes, characterized by Eomes upregulation in the absence of T-bet, in line with its recognized role in the generation of thymic innate-like CD8+ T cells. The clinical relevance of these findings is further supported by the profile of IL-4 production and IL-4 receptor expression that we identified in the human thymus. Importantly, human cord blood CD4+ T cells preserve the ability to generate Eomes+ CD8+ T cells in the presence of IL-4, with implications in neonatal immunity. Our results support a role for IL-4 in the dynamic regulation of human thymocyte plasticity and identify novel strategies to modulate immune responses.

Antenatal Steroids and Cord Blood T-cell Glucocorticoid Receptor DNA Methylation and Exon 1 Splicing.

Antenatal administration of glucocorticoids such as betamethasone (BMZ) during the late preterm period improves neonatal respiratory outcomes. However, glucocorticoids may elicit programming effects on immune function and gene regulation. Here, we test the hypothesis that exposure to antenatal BMZ alters cord blood immune cell composition in association with altered DNA methylation and alternatively expressed Exon 1 transcripts of the glucocorticoid receptor (GR) gene in cord blood CD4+ T-cells. Cord blood was collected from 51 subjects in the Antenatal Late Preterm Steroids Trial: 27 BMZ, 24 placebo. Proportions of leukocytes were compared between BMZ and placebo. In CD4+ T-cells, methylation at CpG sites in the GR promoter regions and expression of GR mRNA exon 1 variants were compared between BMZ and placebo. BMZ was associated with an increase in granulocytes (51.6% vs. 44.7% p = 0.03) and a decrease in lymphocytes (36.8% vs. 43.0% p = 0.04) as a percent of the leukocyte population vs. placebo. Neither GR methylation nor exon 1 transcript levels differed between groups. BMZ is associated with altered cord blood leukocyte proportions, although no associated alterations in GR methylation were observed.

Human Umbilical Cord Blood-Derived CD133+CD34+ Cells Protect Retinal Endothelial Cells and Ganglion Cells in X-Irradiated Rats through Angioprotective and Neurotrophic Factors.

Radiation retinopathy (RR) is a common complication following radiation therapy of globe, head, and neck malignancies, and is characterized by microangiopathy, neuroretinopathy, and the irreversible loss of visual function. To date, there is no effective treatment for RR. Stem cells have been clinically used to treat retinal degeneration. CD133+CD34+ cells from human umbilical cord blood (hUCB-CD133+CD34+ cells), a subpopulation of hematopoietic stem cells, were applied to determine their protective efficacy on irradiated rat retinas. After X-ray irradiation on the retinas, rats were intravitreally injected with hUCB-CD133+CD34+ cells. Transplantation of hUCB-CD133+CD34+ cells prevented retinal dysfunction 2 weeks post-operation and lasted at least 8 weeks. CD133+CD34+ cells were distributed along the retinal vessel and migrated to the ganglion cell layer. Moreover, grafted CD133+CD34+ cells reduced the apoptosis of endothelial and ganglion cells in irradiated rats and increased the number of survived CD31+ retinal endothelial cells and Brn3a+ ganglion cells at 2 and 4 weeks, respectively, post-operation. Co-culturing of CD133+CD34+ cells or supernatants with irradiated human retinal microvascular endothelial cells (hRECs) in vitro, confirmed that CD133+CD34+ cells ameliorated hREC apoptosis caused by irradiation. Mechanistically, we found that angioprotective mediators and neurotrophic factors were secreted by CD133+CD34+ cells, which might attenuate irradiation-induced injury of retinal endothelial cells and ganglion cells. hUCB-CD133+CD34+ cell transplantation, as a novel treatment, protects retinal endothelial and ganglion cells of X-irradiated rat retinas, possibly through angioprotective and neurotrophic factors.

Effect of expansion of human umbilical cord blood CD34\u2009+\u2009cells on neurotrophic and angiogenic factor expression and function

The use of CD34 + cell-based therapies has largely been focused on haematological conditions. However, there is increasing evidence that umbilical cord blood (UCB) CD34 + -derived cells have neuroregenerative properties. Due to low cell numbers of CD34 + cells present in UCB, expansion is required to produce sufficient cells for therapeutic purposes, especially in adults or when frequent applications are required. However, it is not known whether expansion of CD34 + cells has an impact on their function and neuroregenerative capacity. We addressed this knowledge gap in this study, via expansion of UCB-derived CD34 + cells using combinations of LDL, UM171 and SR-1 to yield large numbers of cells and then tested their functionality. CD34 + cells expanded for 14 days in media containing UM171 and SR-1 resulted in over 1000-fold expansion. The expanded cells showed an up-regulation of the neurotrophic factor genes BDNF, GDNF, NTF-3 and NTF-4, as well as the angiogenic factors VEGF and ANG. In vitro functionality testing showed that these expanded cells promoted angiogenesis and, in brain glial cells, promoted cell proliferation and reduced production of reactive oxygen species (ROS) during oxidative stress. Collectively, this study showed that our 14-day expansion protocol provided a robust expansion that could produce enough cells for therapeutic purposes. These expanded cells, when tested in in vitro, maintained functionality as demonstrated through promotion of cell proliferation, attenuation of ROS production caused by oxidative stress and promotion of angiogenesis.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.

Reduced Plasmacytoid Dendritic Cell Output Is Associated With High Risk in Low-grade Myelodysplastic Syndrome.