Umbilical Cord Blood CD34 Research Articles

ENGRAFTMENT POTENTIAL OF CD34+ CELLS FROM SEVERE APLASTIC ANEMIA PATIENTS EXPANDED WITH UM171

Immune aplastic anemia (AA) is a hematological disease characterized by pancytopenia and bone marrow (BM) hypocellularity. It is caused by an immune attack against hematopoietic stem and progenitor cells (HSPC). Its treatment is based on allogeneic BM transplantation and immunosuppressive therapy. More recently, the addition of eltrombopag showed that the expansion of hematopoietic precursors is a therapeutic alternative. Fares et al. demonstrated that the small molecule UM171 is capable of expanding the most primitive CD34 + cell compartment of umbilical cord blood (UCB) ex vivo. Our research group showed that UM171 is also capable of effectively expanding immature subpopulations of CD34 + cells from patients with immune AA. The aim of this study was to evaluate the engraftment capacity of CD34 + cells from patients with AA expanded with UM171 in a murine xenotransplantation model. CD34 + cells were purified from either bone marrow of one patient or one UCB unit using immunomagnetic labeling with human CD34 MicroBeads, MS columns, and a magnetic separator. Cells were cultured for seven days in medium supplemented with cytokines and 35 nM UM171 or 0.1% DMSO (control) and then transplanted via retro-orbital injection into pre-conditioned 8-to-12-week-old female NSG-SGM3 (NSGS) mice. The progeny of 3×10 4 UCB CD34 + cells or 8.250 AA patient CD34 + cells was transplanted per mouse (four mice per experimental condition). The conditioning regimen consisted of two intraperitoneal injections of 12.5 mg/kg busulfan administered 48 and 24 hours before transplantation. Human hematopoietic cells were monitored in the murine BM by flow cytometry at week 20 post-transplantation. BM cells were collected from the two femurs and the two tibias of each animal and treated twice with red cell lysis, labeled with APC-Cy7 anti-mouse and PerCP-Cy5.5 anti-human CD45 antibodies, then analyzed on a flow cytometer using the FlowJo V.10 software. We found that both progenies of UCB and AA BM CD34 + cells cultured with UM171 showed an improved reconstitution potential in mice BM 20 weeks after transplant compared to DMSO, which was demonstrated by the detection of higher percentages of human CD45 + cells in the mice BM (UCB: UM171 = 10,07% ±2,29 vs DMSO = 3,73% ±1,99; AA BM: UM171 = 0,095% ±0,075 vs DMSO = 0,042% ±0,002; data represent mean ±Standard Error). Differences were not significant. We wondered whether the UM171 treatment would impact the engraftment capacity of the in vitro expanded CD34 + cells. The enhanced engraftment capacity enabled by UM171 could allow for immune AA patients to be treated with a novel strategy, autologous transplantation of their own CD34 + expanded cells. The present study indicates that UM171 could be a promising candidate drug for treating patients with immune AA. More replicates (patients and controls) are required to firmly establish the benefit of UM171 in conferring repopulation potential to ex vivo expanded CD34 + cells from AA patients. In initial experiments, in vitro treatment with UM171 appears to increase the engraftment of CD34 + cells isolated from UCB and from the BM of immune AA patients. These results need to be confirmed in additional experiments.

Treg in GVHD Prophylaxis: Unique Transcription Factor Regulation of Foxp3 Expression in Itreg Derived from Human Umbilical Cord Blood (UCB) Vs Adult (AB) Naive CD4 T Cells

Early clinical experience identifies third party allogeneic natural T regulatory cells (nTreg) to be safe and effective in acute GVHD (aGVHD) prophylaxis (CG Brunstein et al. 2016). However the low proliferative potential of nTreg remains a challenge to broader clinical application. Inducible Treg (iTreg) can reestablish tolerance in settings where Tregs are decreased or defective. However instability of expression of Transcription factor Fork head box P3 (Foxp3), essential for CD4+ iTreg induction and function, also poses a significant barrier to successful clinical application. Further understanding of Foxp3 transcription factor partners and their function is emerging. Foxp3 is known to regulate Treg function through cooperation with Nuclear Factor of Activated T-Cells (NFAT1). More recently, broad complex-Tramtrack-Bric-a-brac domain (BTB) and Cap'n'collar (CNC) homology 1, basic leucine zipper transcription factor 2 (BACH2) has been shown to repress effector programs to stabilize Treg-mediated immune homeostasis in murine KO studies (Roychoudhuri R. 2013). However, little is known in normal human CD4 T cells what role, if any, BACH2 may play in Foxp3+ regulation. Previous work by this group identified higher BACH2 expression in UCB vs. AB CD4+ T cells, and further that BACH2 regulates expression of IL-2 in UCB CD4+ T-cells lacking normal expression of NFAT1 protein (M. Lesniewski et al. 2008). In our current studies, we compared BACH2 function in Foxp3+ iTreg derived from UCB vs. AB naïve CD4+CD45RA+ cells. We observe that: i.) naïve UCB CD4+ T cells highly express BACH2, 21-fold higher compared to AB CD4+ T cells (including protein and mRNA expression (n=6; p<0.01), ii.) Foxp3+ expression is 2.5-fold higher in iTreg derived from UCB CD4+CD45RA+ T cells vs. AB (86.8 ± 3.13 vs. 34.3 ± 4.2, n=12; p<0.001), iii) absolute number of Foxp3+ iTreg generated from UCB vs. AB CD4+CD45RA+ T cells is 4-fold higher (3.8 x 106 vs. 1.0 x 106), in standard iTreg differentiation conditions, e.g. CD2/3/2/28 stimulation in X-VIVO15 media with 5 ng/ml transforming growth factor # (TGF#, iv.) suppressive function of UCB Foxp3+ iTreg is more potent in mixed lymphocyte culture compared to AB Foxp3+ iTreg (89% vs. 58% inhibition of T cell proliferation in 1:1 ratio, n=9; p<0.001), v.) UCB iTreg exhibit higher surface PD-1 and CTLA-4 MFI expression (PD-1: UCB: 2835 vs. AB: 1398; p<0.01, CTLA-4: UCB: 1558 vs. AB: 653; p<0.001, n=9). Loss of BACH2 expression in UCB CD4+ T cells results in 42% loss of Foxp3 expression by FACS (Empty shRNA: 78.8% vs. Bach2 shRNA: 46.2%, n=7; p<0.001) in transient shRNA knock-down experiments. Cross-linking chromatin immunoprecipitation (ChIP) showed BACH2 binding to the Foxp3 promoter in UCB iTreg but not AB iTreg (n=2, *; p<0.01) (Figure 1A). BACH2 inhibition via shRNA knockdown notably results in 36-fold reduction of Foxp3 mRNA expression (n=4, *; p<0.01) and 16-fold reduction of Foxp3 down-stream CTLA-4 (n=4, *; p<0.01) by qPCR (Figure 1B). Collectively, these data support the hypothesis that BACH2 is an important transcription factor partner that serves to stabilize Foxp3+ inducible regulatory T cells derived from UCB CD4+ naïve T cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bioactive SL-13R Peptide Expands Human Umbilical Cord Blood Hematopoietic Stem Cells and Maintains Long-Term Reconstitution Ability

Hematopoietic stem cell (HSC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSCs, the presence of an insufficient number of HSCs in these preparations limits their use, prompting need for ex vivo HSC amplification. To establish a more efficient method to expand UCB HSCs, we developed the SL-13R peptide and cultured UCB CD34+ cells with peptide in serum-free medium containing a cytokine cocktail. Following 9 days of culture with peptide, the number of total cells, CD34+CD38- cells and hematopoietic colonies significantly increased relative to control cells grown without peptide. Transplantation of these cells into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution ability. To understand how the peptide promotes HSC expansion, we identified PLEC and ERLIN2 proteins as peptide interactors and undertook loss-of-function analysis to determine whether either was required for peptide function in our culture system. PLEC knockdown UCB CD34+ cells cultured with peptide showed a decreased number of hematopoietic colonies relative to peptide-treated, non-knockdown controls. By contrast, ERLIN2 knockdown had little effect in the presence of peptide. This work suggests that PLEC functions in HSC expansion promoted by SL-13R. In summary, we have identified a novel peptide promoting expansion of UCB CD34+ cells with long-term reconstitution ability. Its use may facilitate clinical use of UCB HSCs. DisclosuresSugiyama:Science Lustre, Ltd: Equity Ownership.

Evaluation of the Efficacy and Safety of Treatment on Using Single-Unit Unrelated Umbilical Cord Blood Transplantation Based on Umbilical Cord Blood Provided By China's Public Cord Blood Bank

Objective It mainly analyzes the umbilical cord blood used in umbilical cord blood transplantation (UCBT), which is provided by China's public cord blood bank. It made an analysis for the effect and safety of malignant hematologic diseases cured by UCBT. Methods We retrospectively analyzed 353 cases of patients with malignant hematologic diseases who accepted myeloablative single-unit unrelated donor UCBT without ATG at our center between Jan 2009 and December 2016. It made a comparative analysis for the umbilical cord blood's TNC before freezing and after thawing, the cells of CD34+, the recovery rate of cells and the clinical effect of UCBT. Results 353 units of umbilical cord blood used in UCBT respectively came from eight China's public cord blood banks. The average of 353 units of umbilical cord blood's TNC before freezing、after the tubular recovery、transfusion were respectively 18.4×108、16.27×108、15.75×108 , the number of TNC was significantly reduced after the recovery of the tubule and transfusion(P=0.000),and there was no difference for the tubular recovery and transfusion(P=0.132) . The average of CD34+ cells before freezing and after the tubular recovery were 11.16×106, 8.68×106 (P=0.000). The average recovery rate of the tubular recovery 、 umbilical cord blood transfusion and CD34+ cells after the tubular recovery was respectively 88.5%、86.5%、80.3%, the difference of recovery rate in each bank was obvious statistically significant (P=0.000,P=0.000,P=0.000). When 353 patients made the UCBT, the cumulative incidence of neutrophil 、platelet、erythrocyte engraftment was respectively 95.8%、84.9%、89.0%, it had the engraftment median time was respectively 17d (11d-42d)、38d (14d-120d)、23d (9d-60d). After CB provided by each bank was used in UCBT, it got the difference about cumulative incidence of engraftment (P=0.025,P=0.0045,P=0.00072). During 100 days after UCBT, the cumulative incidence about the Ⅱ-Ⅳ degrees of acute graft-versus-host disease (aGVHD) were 46.5%. After CB provided by each bank was used in UCBT, it got the no difference about cumulative incidence of aGVHD (P=0.904). During 3 years after UCBT, the cumulative incidence of relapse was 17.85%. After CB provided by each bank was used in UCBT, the difference about cumulative incidence of relapse was no statistical significance (P=0.654). During 3 years after UCBT, the overall survival (OS) rate、disease free survival (DFS) rate and the rate of graft-versus-host diseases/relapse-free survival (GRFS) were respectively71.1 %、66%、59.7%. After CB provided by each bank was used in UCBT, the difference about the rate of OS、DFS、GRFS were no statistical significance(P=0.689,P=0.719,P=0.477). Conclusion The umbilical cord blood provided by China's public cord blood bank was used in UCBT. It has a high safety and good efficacy in treating malignant hematologic diseases. But it needs to set up the standardized and normalized quality-control system of umbilical cord blood for China's public cord blood bank. DisclosuresNo relevant conflicts of interest to declare.

Microrna-15a-5p Regulates Multiple Components of GPVI-Mediated Platelet and Megakaryocyte Activation Pathway

Introduction: Platelet activation by collagen is a major effector ofnormal hemostasis. Mutations in GPVI, the major collagen signaling receptor, cause platelet-type bleeding. Two single nucleotide polymorphisms in GPVI have been identified as risk factors for myocardial infarction. Elevated levels of GPVI have been reported in patients with coronary syndromes. GPVI and its signaling pathway components for platelet activation have become potential antithrombotic targets. Reports have indicated microRNAs (miRs) regulate platelet activation, but none have considered the mechanisms involving miR-mRNA associations regulating the GPVI-mediated platelet activation pathway.Aims: To identify and characterize miRs regulating GPVI-mediated megakaryocyte (MK) integrin activation.Methods : Candidate miRs were identified via a platelet function association study. miR function was assessed by knockdown or overexpression studies in MKs derived from umbilical cord blood (CB) CD34+ stem cells. MK αIIbβ3 activation was measured by flow cytometric quantification of PAC-1 binding after collagen-related peptide (CRP) stimulation. miR and mRNA quantification were performed using quantitative polymerase chain reaction.Results: Our P latelet R NA A nd e X pression (PRAX1) study identified 20 miRs associated with CRP-induced platelet aggregation (p<0.008; n=154). To minimize false positives, we considered only miRs expressed in both platelets and cultured MKs, reducing the potential candidates to 10. We focused on miR-15a-5p because it is negatively correlated with platelet activation through CRP (p=0.0075) (Fig 1A) and because miR-15a-5p targets BCL2, which is known to enhance CRP-induced platelet activation. miR-15a-5p was not found to be associated with platelet aggregation in response to arachidonic acid, ADP, PAR1-AP or PAR4-AP, suggesting specificity of miR-15a-5p for GPVI-mediated integrin activation pathway. Knock down of miR-15a-5p using locked nucleic acid (LNA) in CBMKs caused a ~38% increase in CRP-induced integrin-activation (p=0.0035; n=7) compared to the scrambled (Scr) LNA control (Fig 1B), presumably by releasing miR-15a-5p repression of activation. Fig 1B shows lentiviral overexpression of miR-15a-5p decreased CRP-induced integrin-activation by ~33% (p=0.0154; n=4) as compared to empty vector (EV) control. To understand how miR-15a-5p regulates integrin activation, we bioinformatically identified putative mRNA targets FYN, SRGN, FCER1G, MYLK and PRKCQ, predicted to be regulated by miR-15a- 5p and known as signaling molecules in the GPVI activation pathway. Of note, GPVI was not predicted to be a target of miR-15a-5p and BCL2 was not included because it has been studied before. Importantly, these target genes are positively associated with CRP-induced platelet aggregation (all p<0.004; n=154). The transcript levels of FYN, FCER1G, MYLK and PRKCQ were derepressed upon miR-15a-5p knockdown (Fig 1C) as compared to Scr LNA (all p<0.05; n>4). In miR-15a-5p overexpressed CBMKs, transcript levels of FYN, FCER1G and PRKCQ were decreased compared to EV (Fig 1D) (all p<0.05; n=4). SRGN only showed trends upon miR-15a-5p manipulation. MYB and ACTIN were used as positive and negative controls, respectively. Initial studies of miR-15a-5p knockdown showed a trend for increased FYN, FCER1G and PRKCQ protein levels (Fig 1E; n=3). miR activity requires binding to Argonaute2 (Ago2), which catalyzes translational repression and mRNA degradation. We demonstrated that Ago2 immunoprecipitation enriches miR-15a-5p and its target FYN, SRGN, FCER1G, MYLK and PRKCQ mRNAs (Fig 1F). These data suggest miR-15a-5p and its targets are processed through the Ago complex and thereby, are potential miR-mRNA pairs together regulating GPVI-mediated integrin activation pathway.Conclusions:miR-15a-5p negatively regulates GPVI-mediated MK integrin activation. miR-15a-5p appears to act as a ‘master-miR’ regulating multiple components of GPVI-mediated platelet and MK activation. As miRs are increasingly becoming clinical therapeutic targets, miR-15a-5p overexpression shows potential for treating thrombosis, whereas its inhibition could be effective in bleeding disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Lnk/Sh2b3 Deficiency Restores Hematopoietic Stem Cell Function and Genome Integrity in Fanconi Anemia

▪Although initially identified over 85 years ago, Fanconi Anemia (FA) remains a fatal genetic disease. Patients with FA exhibit developmental malformations, bone marrow failure, and increased cancer susceptibility. Twenty-one FA genes cooperate in a genome stability pathway that is essential for repair of DNA interstrand crosslinks (ICLs) and attenuation of replication stress. Cells derived from FA patients are hypersensitive to ICL-inducing agents such as Mitomycin C (MMC), a hallmark of FA. In addition, FA hematopoietic stem and progenitor cells (HSPCs) show growth retardation and increased apoptosis culminating in hematopoietic stem cell (HSC) exhaustion and bone marrow failure. The poor growth of HSPCs also poses a significant obstacle in gene therapy for FA patients. Moreover, unrepaired DNA damage increases genome instability and promotes leukemia and cancer. Of note, interventions to mitigate HSPC defects in FA do not exist, aside from allogeneic bone marrow transplantation.Remarkably, we reveal here that loss of the adaptor protein Lnk (also called Sh2b3) suppresses FA associated phenotypes without further accelerating neoplastic transformation. LNK is a negative regulator of HSC self-renewal in part by attenuating cytokine signaling, in particular thrombopoietin (TPO)-activated JAK2 signaling in HSCs. Fancd2-/-;Lnk-/- mice exhibit increased phenotypic HSCs in comparison to wildtype (WT) animals. The reconstituting ability of Fancd2-/-;Lnk-/- HSCs is also largely restored to WT levels in serial transplantation assays. In addition, Lnk deficiency restored the growth and survival of FA HSPCs in ex vivo culture. More importantly, Lnk deficiency mitigated chromosomal aberrations associated with FA upon proliferation. To determine if Lnk deficiency mitigates ICL repair, we examined MMC sensitivity in primary HSPCs that were dually null for Lnk and FancD2. Our data suggest that LNK does not play an overt role in ICL repair. Rather, Lnk deficiency in HSPCs mitigated replication stress by stabilizing replication forks and reduces genome instability associated with FA. Hence, our data suggest that Lnk deficiency alleviates replication stress-induced DNA damage and genome instability, thereby restoring cell proliferation and survival of Fancd2-/- HSPCs ex vivo and HSC functions in vivo.Last and importantly, we began to test if LNK inhibition restores human FA HSPCs. We first generated FA-like HSPCs by knocking down FANCD2 in normal umbilical cord blood (UCB) CD34+ cells. Our results demonstrated that shRNA-mediated depletion of LNK in human CD34+ cells rescued progenitor cell numbers and growth in FA-like HSPCs. Take together, our findings reveal a previously unappreciated role of cytokine/JAK signaling in alleviating replication stress and promoting replication-associated genome maintenance. Our studies also illuminate replication stress as a major underlying origin of bone marrow failure in FA patients and have implications for therapeutic strategies to treat FA associated bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Short-term exposure of umbilical cord blood CD34+ cells to human platelet lysate and cytokines enhances engraftment

Short-term exposure of umbilical cord blood CD34+ cells to human platelet lysate and cytokines enhances engraftment

The Bioactive Peptide SL-13R Expands Human Umbilical Cord Blood Hematopoietic Stem and Progenitor Cells In Vitro.

Hematopoietic stem and progenitor cell (HSPC) transplantation is a curative treatment of hematological disorders that has been utilized for several decades. Although umbilical cord blood (UCB) is a promising source of HSPCs, the low dose of HSPCs in these preparations limits their use, prompting need for ex vivo HSPC expansion. To establish a more efficient method to expand UCB HSPCs, we developed the bioactive peptide named SL-13R and cultured UCB HSPCs (CD34+ cells) with SL-13R in animal component-free medium containing a cytokine cocktail. Following 9 days of culture with SL-13R, the numbers of total cells, CD34+, CD38− cells, and hematopoietic stem cell (HSC)-enriched cells were significantly increased relative to control. Transplantation of cells cultured with SL-13R into immunodeficient NOD/Shi-scid/IL-2Rγ knockout mice confirmed that they possess long-term reconstitution and self-renewal ability. AHNAK, ANXA2, and PLEC all interact with SL-13R. Knockdown of these genes in UCB CD34+ cells resulted in reduced numbers of hematopoietic colonies relative to SL-13R-treated and non-knockdown controls. In summary, we have identified a novel bioactive peptide SL-13R promoting expansion of UCB CD34+ cells with long-term reconstitution and self-renewal ability, suggesting its clinical use in the future.

Humanized NOD/SCID/IL2rγnull mice exhibit functionally augmented human regulatory T cells associated with enzymatic up-regulation of H3K27me3 in comparison with humans.

Humanized non-obese diabetic/severe combined immunodeficiency/interleukin-2 receptor-γ-null (NOD/SCID/IL2rγnull ) [humanized (huNSG)] mice engrafted with human hematopoietic cells have been used for investigations of the human immune system. However, the epigenetic features of the human regulatory T (Treg ) cells of huNSG mice have not been studied. The objective of this study was to clarify the characteristics of human Treg cells in huNSG mice, especially in terms of the epigenetic aspects. We compared the populations, inhibitory molecule expression and suppressive capacity of human Treg cells in spleens harvested from the huNSG mice 120days after the engraftment of human umbilical cord blood CD34+ cells with human peripheral blood mononuclear cells (PBMCs). Histone modifications and enhancer of zeste homolog 2 (Ezh2), an H3K27 methyltransferase, of human Treg cells were quantified in huNSG mice and human PBMCs. The effect of Ezh2 inhibitor on human Treg cells exposed to interleukin (IL)-6 was also compared between them. Human Treg cells in the spleens of huNSG mice showed an increased proportion among CD4+ T cells, higher expressions of forkhead box protein 3 (FoxP3), cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor-related protein (GITR), a higher production of IL-10 and enhanced suppressive capacity when compared with those in human PBMCs. H3K27me3 and Ezh2 were specifically up-regulated in human Treg cells of huNSG mice in comparison with those of human PBMCs. The decrease in Treg cells induced by IL-6 exposure was attenuated in huNSG mice when compared with human PBMCs, while the difference between them was cancelled by addition of Ezh2 inhibitor. In conclusion, huNSG mice exhibit functionally augmented human Treg cells owing to enzymatic up-regulation of H3K27me3.

Umbilical cord blood CD34+ cells administration improved neurobehavioral status and alleviated brain injury in a mouse model of cerebral palsy

PurposeCerebral palsy (CP) is the most common neuromuscular disease in children, and currently, there is no cure. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment for CP. However, these studies either examined the effects of UCB cell fraction with a short experimental period or used neonatal rat models for a long-term study which displayed an insufficient immunological reaction and clearance of human stem cells. Here, we developed a CP model by hypoxia-ischemic injury (HI) using immunodeficient mice and examined the effects of human UCB CD34+ hematopoietic stem cells (HSCs) on CP therapy over a period of 8 weeks.MethodsSixty postnatal day-9 (P9) mouse pups were randomly divided into 4 groups (n = 15/group) as follows: (1) sham operation (control group), (2) HI-induced CP model, (3) CP model with CD34+ HSC transplantation, and (4) CP model with CD34- cell transplantation. Eight weeks after insult, the sensorimotor performance was analyzed by rotarod treadmill, gait dynamic, and open field assays. The pathological changes in brain tissue of mice were determined by HE staining, Nissl staining, and MBP immunohistochemistry of the hippocampus in the mice.ResultsHI brain injury in mice pups resulted in significant behavioral deficits and loss of neurons. Both CD34+ HSCs and CD34- cells improved the neurobehavioral statuses and alleviated the pathological brain injury. In comparison with CD34- cells, the CD34+ HSC compartments were more effective.ConclusionThese findings indicate that CD34+ HSC transplantation was neuroprotective in neonatal mice and could be an effective therapy for CP.

Allogeneic human umbilical cord Wharton\u2019s jelly stem cells increase several-fold the expansion of human cord blood CD34+ cells both in vitro and in vivo

BackgroundThe transplantation of human umbilical cord blood (UCB) CD34+ cells has been successfully used to treat hematological disorders but one major limitation has been the low cell numbers available. Mesenchymal stem cells (MSCs) lying within the bone marrow in vivo behave like a scaffold on which CD34+ cells interact and proliferate. We therefore evaluated the use of allogeneic MSCs from the human UC Wharton’s jelly (hWJSCs) as stromal support for the ex vivo expansion of CD34+ cells.MethodsWe performed an in-depth evaluation of the primitiveness, migration, adhesion, maturation, mitochondrial behavior, and pathway mechanisms of this platform using conventional assays followed by the evaluation of engraftment potential of the expanded CD34+ cells in an in vivo murine model.ResultsWe demonstrate that hWJSCs and its conditioned medium (hWJSC-CM) support the production of significantly high fold changes of CD34+, CD34+CD133+, CD34+CD90+, CD34+ALDH+, CD34+CD45+, and CD34+CD49f+ cells after 7 days of interaction when compared to controls. In the presence of hWJSCs or hWJSC-CM, the CD34+ cells produced significantly more primitive CFU-GEMM colonies, HoxB4, and HoxA9 gene expression and lower percentages of CD34+CXCR4+ cells. There were also significantly higher N-cadherin+ cell numbers and increased cell migration in transwell migration assays. The CD34+ cells expanded with hWJSCs had significantly lower mitochondrial mass, mitochondrial membrane potential, and oxidative stress. Green Mitotracker-tagged mitochondria from CD34+ cells were observed lying within red CellTracker-tagged hWJSCs under confocal microscopy indicating mitochondrial transfer via tunneling nanotubes. CD34+ cells expanded with hWJSCs and hWJSC-CM showed significantly reduced oxidative phosphorylation (ATP6VIH and NDUFA10) and increased glycolytic (HIF-1a and HK-1) pathway-related gene expression. CD34+ cells expanded with hWJSCs for 7 days showed significant greater CD45+ cell chimerism in the bone marrow of primary and secondary irradiated mice when transplanted intravenously.ConclusionsIn this report, we confirmed that allogeneic hWJSCs provide an attractive platform for the ex vivo expansion of high fold numbers of UCB CD34+ cells while keeping them primitive. Allogeneic hWJSCs are readily available in abundance from discarded UCs, can be easily frozen in cord blood banks, thawed, and then used as a platform for UCB-HSC expansion if numbers are inadequate.

Human CD133-positive hematopoietic progenitor cells enhance the malignancy of breast cancer cells

BackgroundHuman CD133+ hematopoietic progenitor cells (HPCs) are a specific subset of cells that can regulate tumor malignancy. However, the mechanism by which CD133+ HPCs affect the malignancy of human breast cancer has not been reported.MethodsCD133+ HPCs were isolated and purified from human umbilical cord blood (UCB). We used in vitro culture of MCF-7 and MDA-MB-231 cell lines, and MCF-7 and MDA-MB-231 cells in nude mice to evaluate whether CD133+ HPCs affected the apoptosis, proliferation, invasion and epithelial mesenchymal transition EMT of breast cancer cells.ResultsCo-culture with CD133+ HPCs, but not UCB CD133- cells, promoted the proliferation of human breast cancer MCF-7 and MDA-MB-231 cells, accompanied by reducing in vitro spontaneous apoptosis. Co-administration of these two lines with CD133+ HPCs significantly enhanced the growth of implanted breast cancer in vivo. Furthermore, co-culture with CD133+ HPCs, enhanced the invasion of breast cancer cells, N-cadherin and Vimentin expression, but reduced E-cadherin expression in breast cancer cells.ConclusionsOur study demonstrated that CD133+ HPCs enhance the malignancy of breast cancer cells by attenuating spontaneous apoptosis and promoting the process of epithelial mesenchymal transition. These findings may provide new insights into the role of human CD133+ HPCs in breast cancer pathogenesis. Therefore, CD133+ HPCs may be a new therapeutic target for inhibiting the progression of breast cancer.

Plant cell-secreted stem cell factor stimulates expansion and differentiation of hematopoietic stem cells

Ex vivo generation of red blood cells (RBCs) from hematopoietic stem cells (HSCs) used for blood transfusion represents one of the focuses in current regenerative medicine. However, massive production of HSCs-based RBCs requires a significant quantity of erythropoietic growth factors, making manufacturing at large scale cost prohibitive. Plant cell culture is proposed to be a promising bioproduction platform for functional human proteins in a safe and cost-efficient manner. This study exploited a proprietary technology, named HypGP engineering technology, for high-yield production of one of the key erythropoietic growth factors--stem cell factor (SCF)--in plant cell culture. Specifically, a designer hydroxyproline (Hyp)-O-glycosylated peptide (HypGP) comprised of 20 tandem repeats of the “Ser-Pro” motif, or (SP)20, was engineered at either the N-terminus or C-terminus of SCF in tobacco BY-2 cells. The (SP)20 tag dramatically increased the secreted yields of SCF up to 2.5 μg/mL. The (SP)20-tagged SCF showed bioactivity in promoting the proliferation of the TF-1 cell line, although the SCF-(SP)20 was 8.4-fold more potent than the (SP)20-SCF. Both the (SP)20-SCF and SCF-(SP)20 exhibited desired function in stimulating the expansion and differentiation of human umbilical cord blood CD34+ cells towards RBCs.

Coinhibition of activated p38 MAPKα and mTORC1 potentiates stemness maintenance of HSCs from SR1-expanded human cord blood CD34+ cells via inhibition of senescence.

The stemness of ex vivo expanded hematopoietic stem cells (HSCs) is usually compromised by current methods. To explore the failure mechanism of stemness maintenance of human HSCs, which were expanded from human umbilical cord blood (hUCB) CD34+ cells, by differentiation inhibitor Stem Regenin 1 (SR1), an antagonist of aryl hydrocarbon receptor, we investigated the activity of p38 mitogen‐activated protein kinase α (p38 MAPKα, p38α) and mammalian target of rapamycin complex 1 (mTORC1), and their effect on SR1‐expanded hUCB CD34+ cells. Our results showed that cellular senescence occurred in the SR1‐expanded hUCB CD34+ cells in which p38α and mTORC1 were successively activated. Furthermore, their coinhibition resulted in a further decrease in hUCB CD34+ cell senescence without an effect on apoptosis, promoted the maintenance of expanded phenotypic HSCs without differentiation inhibition, increased the hematopoietic reconstitution ability of multiple lineages, and potentiated the long‐term self‐renewal capability of HSCs from SR1‐expanded hUCB CD34+ cells in NOD/Shi‐scid/IL‐2Rγnull mice. Our mechanistic study revealed that senescence inhibition by our strategy was mainly attributed to downregulation of the splicesome, proteasome formation, and pyrimidine metabolism signaling pathways. These results suggest that coinhibition of activated p38α and mTORC1 potentiates stemness maintenance of HSCs from SR1‐expanded hUCB CD34+ cells via senescence inhibition. Thus, we established a new strategy to maintain the stemness of ex vivo differentiation inhibitor‐expanded human HSCs via coinhibition of multiple independent senescence initiating signal pathways. This senescence inhibition‐induced stemness maintenance of ex vivo expanded HSCs could also have an important role in other HSC expansion systems.

Expansion of umbilical cord blood derived haematopoietic stem cells under constant dynamic conditions

Background & Aim Umbilical cord blood (UCB) offers a source of haematopoietic stem and progenitor cells (HSPCs) with a proven clinical rack record. However, one major drawback is the relatively low number of HSPCs within one unit, limiting its clinical potential. Due to this, strategies to expand UCB-HSPCs have been developed and involve the use of additional molecules to support expansion, such as SR-1, UM-171, immobilised Notch ligand Delta-like 1 and nicotinamide. As these potential therapeutics reach late stage clinical trials, considerations for manufacturing in order to meet clinical demand are required. In this study we have investigated the impact that constant shaking, at differing magnitudes, during the expansion of UBC-HSPCs has on the overall total nucleated cell expansion, Lin-CD34+ and Lin-CD34+CD38-CD90+ cell expansion, cell viability and colony forming unit (CFU) potential. Methods, Results & Conclusion Method UCB CD34+ cells were seeded at 1 × 104 cells/mL in IMDM+10% FBS supplemented with 100ng/mL SCF, TPO and Flt3-L and 20ng/mL IL-6 and maintained under static (T-flasks) or shaken (125mL Erlenmeyer flasks, with a 10mm shaking diameter) conditions for nine days. Daily cell counts were completed and immunophenotyping, apoptosis detection and CFU potential were performed on day 0 and day 9. Results UCB-HSPCs were successfully expanded for all donors under both conditions, the final viable cell densities achieved were significantly higher in the shaken conditions compared to the static controls, in addition the final viable cell densities increased with an increasing agitation rate, 3.1±0.3 × 104, 6.2±0.1 × 104, 10.2±0.2 × 104 and 12.4±0.4 × 104 cells mL−1 (mean value ± SD) for static, 30rpm, 70rpm and 110rpm respectively. Immunophenotyping of the expanded cells demonstrated an increase in both Lin-CD34+ HSPCs and Lin-CD34+CD38-CD90+ haematopoietic stem cells, this increase was significantly higher in the shaken conditions compared to the static controls. Finally, the fold change in CFU potential was significantly higher for both granulocyte, monocyte colonies and granulocyte, erythrocyte, monocyte, megakaryocyte colonies compared to static controls and with an increasing agitation rate. Conclusion In this study we have shown that expansion of UCB-HSPCs under shaken conditions improves the overall cellular yield that can be achieved, maintains both Lin-CD34+ and the more primitive Lin-CD34+CD38-CD90+ cell populations and enhances CFU potential.

Heparanase Facilitates PMA-Induced Megakaryocytic Differentiation in K562 Cells via Interleukin 6/STAT3 Pathway.

Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan sulfate and hence participates in remodeling of the extracellular matrix, leading to release of cytokines that are immobilized by binding to heparan sulfate proteoglycans (HSPGs), and consequently activating signaling pathways. This function of HPSE is correlated to its expression level that is normally very low in majority of the tissues. Exceptionally, human platelets express high level of HPSE, suggesting a unique physiological role in this cell. Using K562 cell line, we found a progressive increase of HPSE during the megakaryocytic differentiation. Analysis of a series of megakaryocytic differentiation-related heparin-binding proteins (HBPs) in the cell culture medium revealed an exclusive positive correlation between the level of interleukin 6 (IL-6) and HPSE expression. IL-6 modulated megakaryocytic differentiation through activation of STAT3. Further, we demonstrated that overexpression of HPSE potentiates megakaryocytic differentiation, whereas elimination of HPSE led to a delayed differentiation. This function of HPSE is associated with its activity, as overexpression of inactive HPSE had no effect on IL-6 production and megakaryocytic differentiation. The role of HPSE is further supported by the observation in an umbilical cord blood CD34+ cells megakaryocytic differentiation model. Our data propose a novel role for HPSE in platelets production by a HPSE/IL-6/STAT3 positive feedback loop that specifically regulates megakaryocytes maturation.

Nov/CCN3 Enhances Cord Blood Engraftment by Rapidly Recruiting Latent Human Stem Cell Activity.

SummaryUmbilical cord blood (UCB) has had considerable impact in pediatric stem cell transplantation, but its wider use is limited in part by unit size. Long-term ex vivo culture offers one approach to increase engraftment capacity by seeking to expand stem and progenitor cells. Here, we show brief incubation (8 h) of UCB CD34+ cells with the matricellular regulator Nov (CCN3) increases the frequency of serially transplantable hematopoietic stem cells (HSCs) 6-fold. This rapid response suggests recruitment rather than expansion of stem cells; accordingly, in single-cell assays, Nov increases the clonogenicity of phenotypic HSCs without increasing their number through cell division. Recruitment is associated with both metabolic and transcriptional changes, and tracing of cell divisions demonstrates that the increased clonogenic activity resides within the undivided fraction of cells. Harnessing latent stem cell potential through recruitment-based approaches will inform understanding of stem cell state transitions with implications for translation to the clinic.

Purifying stem cell-derived red blood cells: a high-throughput label-free downstream processing strategy based on microfluidic spiral inertial separation and membrane filtration.

Cell‐based therapeutics, such as in vitro manufactured red blood cells (mRBCs), are different to traditional biopharmaceutical products (the final product being the cells themselves as opposed to biological molecules such as proteins) and that presents a challenge of developing new robust and economically feasible manufacturing processes, especially for sample purification. Current purification technologies have limited throughput, rely on expensive fluorescent or magnetic immunolabeling with a significant (up to 70%) cell loss and quality impairment. To address this challenge, previously characterized mechanical properties of umbilical cord blood CD34+ cells undergoing in vitro erythropoiesis were used to develop an mRBC purification strategy. The approach consists of two main stages: (a) a microfluidic separation using inertial focusing for deformability‐based sorting of enucleated cells (mRBC) from nuclei and nucleated cells resulting in 70% purity and (b) membrane filtration to enhance the purity to 99%. Herein, we propose a new route for high‐throughput (processing millions of cells/min and mls of medium/min) purification process for mRBC, leading to high mRBC purity while maintaining cell integrity and no alterations in their global gene expression profile. Further adaption of this separation approach offers a potential route for processing of a wide range of cellular products.

Impact of maternal and neonatal factors on umbilical cord CD34+ cells.

The achievement of optimal number of CD34+ umbilical cord stem cells is essential for successful umbilical cord stem cell transplantation. So the aim of this study was to assess the potential effect of both maternal and neonatal factors on the umbilical cord blood CD34+ cell count. The study was done on umbilical cord blood samples obtained from 20 mothers during labor. Their ages ranged from 22 to 34 years and were subjected to history taking, physical examination of the baby and assessment of the CD34+ cells count in umbilical cord blood. Number of previous live births and weight of the baby had a significant effect on CD34+ cells count while the sex of the baby, delivery route, maternal age and gestation period had no significant effect on CD34+ cells count. Umbilical cord blood-derived CD34+ cell count is better with good weight and first babies and decreased with subsequent babies.

Humanized Culture Medium for Clinical-Grade Generation of Erythroid Cells from Umbilical Cord Blood CD34+ Cells.

Purpose: Transfusion of red blood cells (RBCs) is a supportive and common treatment in surgical care, trauma, and anemia. However, in vivo production of RBC seems to be a suitable alternative for blood transfusions due to the limitation of blood resources, the possibility of disease transmission, immune reactions, and the presence of rare blood groups. Cell cultures require serum-free or culture media supplemented with highly expensive animal serum, which can transmit xenoviruses. Platelet lysate (PL) can be considered as a suitable alternative containing a high level of growth factors and a low production cost. Methods: Three-step culture media supplemented with PL or fetal bovine serum (FBS) were used for proliferation and differentiation of CD34+ umbilical cord blood stem cells to erythrocytes in co-culture with bone marrow mesenchymal stem cells (BM-MSCs). The cells were cultivated for 15 days and cell proliferation and expansion were assessed using cell counts at different days. Erythroid differentiation genes, CD71 and glycophorin A expression levels were evaluated. Results: Maximum hematopoietic stem cells (HSCs) proliferation was observed on day 15 in PL-containing medium (99±17×103-fold). Gene expression and surface markers showed higher differentiation of cells in PL-containing medium. Conclusion: The results of this study indicate that PL can enhance erythroid proliferation and differentiation of CD34+ HSCs. PL can also be used as a proper alternative for FBS in the culture medium and HSCs differentiation.