Umbilical Cord Blood Hematopoietic Stem Research Articles

The efficacy and safety of third-party umbilical blood/umbilical cord mesenchymal stem cell assisted related haploid hematopoietic stem cell transplantation in pediatric patients with acute leukemia: an observational study.

There is limited data on third-party umbilical cord blood (UCB) or mesenchymal stem cell (MSC) transplantation-assisted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) in pediatric patients. To evaluate the efficacy and safety of UCB and MSC transplantation-assisted haplo-HSCT in pediatric patients with acute leukemia (AL). Observational study. Clinical data of 152 children with AL undergoing haplo-HSCT at the Children's Hospital of Soochow University between January 2020 and June 2022 were collected. The patients were divided into the haplo-HSCT + UCB group (n = 76), haplo-HSCT + MSC group (n = 31), and haplo-HSCT group (n = 45). Hematopoietic reconstruction time, complications within 30 days after transplantation, and survival and recurrence at 3 years after transplantation were compared among the groups. Multivariate analysis revealed that haplo-HSCT with MSC and human leukocyte antigen (HLA) matching ⩾6/10 were independent factors reducing engraftment syndrome (ES) incidence. There were no significant differences among the groups in the hematopoietic reconstruction time or incidence of complications within 30 days after transplantation (p > 0.05). Overall survival, relapse-free survival, cumulative incidence of relapse, cumulative incidence of hematological relapse, and 3-year transplant-related mortality were not significantly different (p > 0.05). The incidence of adverse reactions in the haplo-HSCT + UCB group was 97.3% within 4 h after UCB infusion, with a particularly high occurrence rate of 94.7% for hypertension. No transfusion-related adverse reactions occurred after the transfusion of umbilical cord MSC in the haplo-HSCT + MSC group. MSC-assisted haplo-HSCT can reduce ES incidence after transplantation in pediatric patients with AL. UCB infusion is associated with a high incidence of reversible hypertension. However, no adverse reactions were observed in umbilical cord MSC transfusion.

Effect of cord blood double collection method on cord blood hematopoietic stem cell transplantation-related indices and blood gas analysis.

Umbilical cord blood has been widely used in clinical transplantation. Blood gas analysis of umbilical cord blood is routinely used to evaluate neonatal asphyxia. This study aimed to evaluate an improved umbilical cord blood collection method that does not affect the results of umbilical cord blood gas analysis and hematopoietic stem cell transplantation-related indices. Three hundred pregnant women were recruited between December 2019 and August 2022. In total, 270 umbilical cord blood samples were included and randomly divided into 3 groups. Group A was defined as the group in which both umbilical cord blood samples for hematopoietic stem cell transplantation and blood gas analysis were collected. Group B was defined as the group from which umbilical cord blood was collected for hematopoietic stem cell transplantation. Group C was defined as that wherein umbilical cord blood was collected only for blood gas analysis. Hematopoietic stem cell transplantation-related indices were detected in groups A and B, and blood gas analysis was performed in groups A and C. Hematopoietic stem cell transplantation-related indices were not significantly different between groups A and B. The pH, base excess, and lactic acid values were not significantly different between groups A and C. The cord blood double collection method would not affect the results of umbilical cord blood gas analysis and hematopoietic stem cell transplantation-related indices. It is suitable for cord blood collection when preparing for hematopoietic stem cell transplantation and blood gas analysis.

Vitamin C derivative/AA2P promotes erythroid differentiation by upregulating CA1

Abstract Vitamin C is used to treat anaemia; however, the mechanism through which vitamin C promotes erythroid differentiation is not comprehensively understood. The in vitro erythroid differentiation induction system can reveal the differentiation mechanism and provide erythrocytes for clinical transfusion and anaemia treatment. This process can be promoted by adding small-molecule compounds. In this study, we added l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P), a derivative of vitamin C, to an erythroid differentiation system induced from umbilical cord blood haematopoietic stem and progenitor cells in vitro and detected its effect on erythroid differentiation using single-cell transcription sequencing technology combined with non-targeted metabolism detection. AA2P increased the proportion of late basophilic erythroblasts, upregulating the expression of erythroid-related regulatory molecules GATA1, KLF1, ALAS2, and the globins HBG and HBB. CA1 is a target gene of AA2P, and CA1 knockdown affected the expression of globin-related genes. AA2P also increased glycolysis and decreased oxidative phosphorylation to facilitate terminal erythroid differentiation and enhanced the proliferation of early erythroid progenitors by altering the cell cycle. These results provide a reliable basis for using vitamin C to improve the efficiency of erythropoiesis in vitro and for the clinical treatment of anaemia.

Evaluation of DNA Damage and Repair In In Vitro Expanded Cord Blood CD 34 Positive Hematopoietic Stem Cell

Background and Aims: The occurrence of single and double-strand breaks of DNA damage is the major obstacle for proliferation under various environmental factors and, if not repaired, can result in many consequences, including mutation, cell death, and others. So, the present study was conducted to evaluate the damage of DNA and the expression status of DNA repair system genes before and after stem cell proliferation.
 Materials and Methods: The MACS method isolated the umbilical cord blood hematopoietic stem cells (UCB-HSCs). In order to investigate cell death, the study of Annexin V/PI was done by flow cytometry. Comet assay made observation and identification of DNA breaks, and the expression of genes normally involved in the repair of DNA breaks was evaluated by real-time polymerase chain reaction.
 Results: The average number of stem cells increased by 1.9-fold after three days of proliferation. The apoptotic percentage of cells was negligible (less than 0.2%), and the purity of the CD34+ cells was reduced by about one-third in three days (67%). By examining the expression of DNA repair genes, including KU70, KU80, RAD51, and XRCC1, their increased fold change was not significant. In a microscopic examination of stem cells in the comet assay, there was no significant difference between DNA damage before (1.33% ± 0.31) and after (2.08% ± 0.92) replication.
 Conclusion: In our investigation, neither DNA damage nor changes in the DNA break repair were observed. However, further studies are required to clarify the DNA break repair by recruiting more UCB-HSCs samples.

Cord Blood Plasma and Placental Mesenchymal Stem Cells-Derived Exosomes Increase Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells While Maintaining Their Stemness.

Mesenchymal stem cells (MSCs) have been used for ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) to maintain their primitive characters and long-term reconstitution abilities during transplantation. Therapeutic effects of MSCs mainly rely on paracrine mechanisms, including secretion of exosomes (Exos). The objective of this study was to examine the effect of cord blood plasma (CBP)-derived Exos (CBP Exos) and Placental MSCs-derived Exos (MSCs Exos) on the expansion of UCB HSCs to increase their numbers and keep their primitive characteristics. CD34+ cells were isolated from UCB, cultured for 10 days, and the expanded HSCs were sub-cultured in semisolid methylcellulose media for primitive colony forming units (CFUs) assay. MSCs were cultured from placental chorionic plates. CBP Exos and MSCs Exos compared with the control group significantly increased the number of total nucleated cells (TNCs), invitro expansion of CD34+ cells, primitive subpopulations of CD34+38+ and CD34+38-Lin- cells (p < 0.001). The expanded cells showed a significantly higher number of total CFUs in the Exos groups (p < 0.01). CBP- and placental-derived exosomes are associated with significant ex vivo expansion of UCB HSCs, while maintaining their primitive characters and may eliminate the need for transplantation of an additional unit of UCB.

The impacts of total body irradiation on umbilical cord blood hematopoietic stem cell transplantation.

Umbilical cord blood hematopoietic stem cells are commonly used for hematopoietic system reconstitution in recipients after umbilical cord blood transplantation (UCBT). However, the optimal conditioning regimen for UCBT remains a topic of debate. The exact impact of total body irradiation (TBI) as a part of conditioning regimens remains unknown. The aim of this study was to evaluate the impacts of TBI on UCBT outcomes. This was a multi-institution retrospective study. A retrospective analysis was conducted on the outcomes of 136 patients receiving UCBT. Sixty-nine patients received myeloablative conditioning (MAC), in which 33 underwent TBI and 36 did not, and 67 patients received reduced-intensity conditioning (RIC), in which 43 underwent TBI and 24 did not. Univariate and multivariate analyses were conducted to compare the outcomes and the post-transplant complications between patients who did and did not undergo TBI in the MAC subgroup and RIC subgroup, respectively. In the RIC subgroup, patients who underwent TBI had superior overall survival (adjusted hazard ratio [aHR] = 0.25, 95% confidence interval [CI]: 0.09-0.66, p = 0.005) and progression-free survival (aHR = 0.26, 95% CI: 0.10-0.66, p = 0.005). However, in the MAC subgroup, there were no statistically significant differences between those receiving and not receiving TBI. In the setting of RIC in UCBT, TBI utilization can improve overall survival and progression-free survival. However, TBI does not show superiority in the MAC setting.

Comparison of Maturation Stages of Natural Killer Cell Differentiation Culture from Cultured and Freshly Isolated Umbilical Cord Blood Hematopoietic Stem Cells

Comparison of Maturation Stages of Natural Killer Cell Differentiation Culture from Cultured and Freshly Isolated Umbilical Cord Blood Hematopoietic Stem Cells

BML-260 promotes the growth of cord blood and mobilized peripheral blood hematopoietic stem and progenitor cells with improved reconstitution ability.

Hematopoietic stem cells (HSCs), which are multipotent and have the ability to self-renew, are frequently used in the treatment of hematological diseases and cancer. Small molecules that target HSC quiescence regulators could be used for ex vivo expansion of both mobilized peripheral blood (mPB) and umbilical cord blood (UCB) hematopoietic stem and progenitor cells (HSPC). We identified and investigated 35 small molecules that target HSC quiescence factors. We looked at how they affected HSC activity, such as expansion, quiescence, multilineage capacity, cycling ability, metabolism, cytotoxicity, and genotoxicity. A transplantation study was carried out on immunocompromised mice to assess the expanded cells' repopulation and engraftment abilities. 4-[(5Z)-5-benzylidene-4-oxo-2-sulfanylidene-1,3-thiazolidin-3-yl]benzoic acid (BML)-260 and tosyl-l-arginine methyl ester (TAME) significantly increased both mPB and UCB-HSPC content and activated HSC re-entry into the cell cycle. The improved multilineage capacity was confirmed by the colony forming unit (CFU) assay. Furthermore, gene expression analysis revealed that BML-260 and TAME molecules aided HSC expansion by modulating cell cycle kinetics, such as p27, SKP2, and CDH1. In addition to these in vitro findings, we discovered that BML-260-expanded HSCs had a high hematopoietic reconstitution capacity with increased immune cell content after xenotransplantation into immunocompromised mice. In addition to the BML-260 molecule, a comparison study of serum-containing and serum-free chemically defined media, including various supplements, was performed. These in vitro and xenotransplantation results show that BML-260 molecules can be used for human HSC expansion and regulation of function. Furthermore, the medium composition discovered may be a novel platform for human HSPC expansion that could be used in clinical trials.

312 - Impacts of Total Body Irradiation in Allogeneic Umbilical Cord Blood Hematopoietic Stem Cell Transplant with Reduced Intensity Conditioning

312 - Impacts of Total Body Irradiation in Allogeneic Umbilical Cord Blood Hematopoietic Stem Cell Transplant with Reduced Intensity Conditioning

Levistilide A Promotes Expansion of Human Umbilical Cord Blood Hematopoietic Stem Cells by Enhancing Antioxidant Activity.

Several approaches to expand human hematopoietic stem cells (hHSCs) clinically along with retainable capability of multipotential differentiation have been reported, but only a few have advanced to evaluation in clinical trials, which limits the application of HSC-based therapy. Here we show a phthalide derivative, Levistilide A (LA), can serve as a promising molecule to expand functional human umbilical cord blood (UCB) HSCs ex vivo. An in-house screen identified LA out of nine natural products as an outstanding candidate for hHSCs expansion. Additionally, our data indicated that LA treatment not only increased the numbers of phenotype-defined HSCs, but also enhanced their colony formation ability. Xenotransplantation assays showed that LA treatment could maintain unaffected engraftment of hHSCs with multilineage differentiation capacity. Further experiments revealed that LA enhanced the antioxidant activity of hHSCs by reducing intracellular and mitochondrial reactive oxygen species (ROS) levels. The identification of LA provides a new strategy in solving the clinical issue of limited numbers of UCB HSCs.

Human-Induced CD49a+ NK Cells Promote Fetal Growth.

CD49a+ natural killer (NK) cells play a critical role in promoting fetal development and maintaining immune tolerance at the maternal-fetal interface during the early stages of pregnancy. However, given their residency in human tissue, thorough studies and clinical applications are difficult to perform. It is still unclear as to how functional human CD49a+ NK cells can be induced to benefit pregnancy outcomes. In this study, we established three no-feeder cell induction systems to induce human CD49a+ NK cells from umbilical cord blood hematopoietic stem cells (HSCs), bone marrow HSCs, and peripheral blood NK cells in vitro. These induced NK cells (iNKs) from three cell induction systems display high levels of CD49a, CD9, CD39, CD151 expression, low levels of CD16 expression, and no obvious cytotoxic capability. They are phenotypically and functionally similar to decidual NK cells. Furthermore, these iNKs display a high expression of growth-promoting factors and proangiogenic factors and can promote fetal growth and improve uterine artery blood flow in a murine pregnancy model in vivo. This research demonstrates the ability of human-induced CD49a+ NK cells to promote fetal growth via three cell induction systems, which could eventually be used to treat patients experiencing adverse pregnancy outcomes.

Effects of bone marrow-derived mesenchymal stem cells exposed to endocrine-disrupting chemicals on the differentiation of umbilical cord blood hematopoietic stem cells.

Endocrine-disrupting chemicals (EDCs), a class of peripheral toxic substances, can cause many environmental and clinical side effects, particularly on the human body's endocrine system. Bisphenol A (BPA) and diethylhexyl phthalate (DEHP) are two well-known EDCs in the medicine industry. However, there are no comprehensive studies on their effects on hematopoiesis. Hence, this study aimed to investigate the effect of these two aforementioned substances on the clonogenic capacity of umbilical cord blood hematopoietic stem cells (CB-HSCs). The HSCs which express CD34 + were isolated from umbilical cord blood by the magnetic-activated cell sorting (MACS) system. To investigate the effects of different optimized concentrations of BPA and DEHP, CB-CD34+ HSCs were exposed to EDCs in semisolid medium. For evaluation of coexposures, CB-CD34+ HSCs were cocultured with bone marrow-derived mesenchymal stem cells (BM-MSCs) in the presence of BPA and DEHP. Finally, the number and types of colonies were evaluated after 14days. Statistical analysis was performed by GraphPad Prism through ANOVA. CB-HSC treated by BPA and DEHP showed a lower absolute colony count than the control group (P < 0.05). Decrease in clonogenic potential of HSCs was more significant in coculture condition by MSCs. In particular, there was a significant decrease in the BFU-E colonies in comedicated-derived fractions (P < 0.0001). In the presence of EDCs such as BPA and DEHP, the patterns of differentiation in CD34+ CB-HSCs changed from suppressed erythroid differentiation toward stimulated myelogenesis pathways.

Hypoxia preconditioned mesenchymal stem cell-derived exosomes induce ex vivo expansion of umbilical cord blood hematopoietic stem cells CD133+ by stimulation of Notch signaling pathway.

Mesenchymal stem cells (MSCs) are crucial cells that play an essential role in the maintenance, self-renewal, and proliferation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) in the bone marrow niche. It has been proven that MSCs can be used as a feeder layer for the proliferation of HSCs to enhance the number of HPCs and HSCs. Recently, it has been demonstrated that MSC-derived exosome (MSC-DE) has critical roles in different biological processes in bone marrow (BM). In the current research, we examined the importance of hypoxia-preconditioned MSC-derived exosomes (HP-MSC-DE) and normoxia-preconditioned MSC-derived exosomes (NP-MSC-DE) in the self-renewal and long-term clonogenic potential of umbilical cord blood hematopoietic stem cells (UCB-HSCs). We showed that the secretion rate and component of the exosome (EXO) were changed in HP-MSC-DE compared to NP-MSC-DE. Notably, the Jagged-1 (Notch ligand) content of EXO was much more plentiful in HP-MSC-DE compared to NP-MSC-DE. The addition of HP-MSC-DE enriched by Jagged-1 to the co-culture system stimulates the Notch pathway on the membrane of UCB-HSCs CD133+ and enhances proliferation. HP-MSC-DE induction using an anti-Jagged-1 antibody suppresses all biological functions of the Jagged-1 protein. Importantly, HP-MSC-DE containing Jagged-1 could change the biology of HSCs CD133+ and increase the self-renewal capacity, quiescence, and clonogenic potential of CD133+ cells. Moreover, they support generating a large number of primitive cells. Our study signified the importance of HP-MSC-DE in the proliferation of UCB-HSCs CD133+, which manifested therapeutic applications of EXO in the enhanced number of HSCs and subsequently alleviated bone marrow transplantation.

Impacts of Total Body Irradiation in Allogeneic Umbilical Cord Blood Hematopoietic Stem Cell Transplantation

Background: Umbilical cord blood hematopoietic stem cell transplant (UCBT) has been practiced as an alternative source of hematopoietic stem cells for patients in need of transplantation. Double-units UCBT has been established as a means of achieving a cell dose of at least 2.5x10 7 nucleated cells per kilogram of body weight in adult recipients. The advantages of UCBT include its rapid availability, reduced stringency in terms of human leukocyte antigen (HLA) match requirements, and subsequent increase in access to transplants for racial minorities. Both related and unrelated UCBTs with single or double units have been performed with high rates of success in both pediatric and adult settings to treat a variety of medical conditions.Prior to undergoing UCBT, recipients must undergo a conditioning regimen to create space in the bone marrow, suppress the immune system to allow for donor stem cell engraftment, and reduce the tumor burden in cases of neoplastic disease. Total body irradiation (TBI) is commonly incorporated into conditioning regimens to enforce these efforts. Although intense myeloablation in general is associated with a lower risk of relapse and graft rejection, greater regimen intensity also leads to a higher rate of transplant related morbidity and mortality (TRM). Inclusion of TBI specifically in conditioning regimens has been shown to result in organ toxicity and subsequent malignant neoplasm. To help mitigate the risks of myeloablative conditioning (MAC) regimens, non-TBI and reduced-intensity conditioning (RIC) regimens have been investigated as a means of reducing TRM and increasing access of transplantation to patients with age disadvantages or significant comorbidities.Despite ongoing investigation, studies comparing conditioning regimens of UCBT, with and without TBI, remain limited. This study, using real-world data collected from 4 institutions, retrospectively analyzed the impact of TBI as part of MAC or RIC conditioning regimens in patients undergoing UCBT.Methods: This is a retrospective study that analyzed the outcomes of 136 patients receiving umbilical cord blood transplants at four institutions. Seventy-nine patients received myeloablative condition (MAC), in which 36 underwent TBI and 33 did not; 67 patients received reduced-intensity condition (RIC), in which 24 underwent TBI and 43 did not. Univariate and multivariate analyses were conducted to compare the outcomes and the post-transplant complications between patients who did and did not undergo TBI in MAC subgroup and RIC subgroup, respectively.Results: Characteristics of UCBT recipients who did and did not undergo TBI, stratified by conditioning regimen were compared with both multivariate and univariate analyses and didn't see significant difference. We didn't observe significant difference in GVHD and transplant-related infection incidence rates between patient subgroup that did and did not undergo TBI as part of their pre-UCBT conditioning regimen via both multivariate and univariate analyses.In RIC subgroup, the patients who underwent TBI appeared to have superior overall survival (adjusted hazard ratio [aHR]=0.25, 95% confidence interval [CI]: 0.09-0.66, p=0.005) (Figure 1), progression-free survival (aHR=0.26, 95% CI: 0.10-0.66, p=0.005) (Figure 2), and shorter time to neutrophil engraftment (aHR=6.26, 95% CI: 2.27 - 17.31, p=0.0004) (Figure 3). However, in MAC subgroup, there were no statistically significant difference between using and not using TBI. There were also no differences between the patients who either underwent or not underwent TBI in terms of acute or chronic GVHD rates or rates of transplant-related infections in both subgroups.Conclusion: Combining with RIC, TBI may improve OS, PFS, and neutrophil engraftment time. However, the incidences of other post-transplant complication were comparable between patients who underwent and did not undergo TBI as part of conditioning regimens during umbilical cord blood transplant. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

'Off-the-Shelf' Immunotherapy: Manufacture of CD8+ T Cells Derived from Hematopoietic Stem Cells.

Cellular immunotherapy is revolutionizing cancer treatment. However, autologous transplants are complex, costly, and limited by the number and quality of T cells that can be isolated from and expanded for re-infusion into each patient. This paper demonstrates a stromal support cell-free in vitro method for the differentiation of T cells from umbilical cord blood hematopoietic stem cells (HSCs). For each single HSC cell input, approximately 5 × 104 T cells were created with an initial five days of HSC expansion and subsequent T cell differentiation over 49 days. When the induced in vitro differentiated T cells were activated by cytokines and anti-CD3/CD28 beads, CD8+ T cell receptor (TCR) γδ+ T cells were preferentially generated and elicited cytotoxic function against ovarian cancer cells in vitro. This process of inducing de novo functional T cells offers a possible strategy to increase T cell yields, simplify manufacturing, and reduce costs with application potential for conversion into chimeric antigen receptor (CAR)-T cells for cancer immunotherapy and for allogeneic transplantation to restore immune competence.

NOTCH-mediated exvivo expansion of human hematopoietic stem and progenitor cells by culture under hypoxia.

SummaryActivation of NOTCH signaling in human hematopoietic stem/progenitor cells (HSPCs) by treatment with an engineered Delta-like ligand (DELTA1ext-IgG [DXI]) has enabled ex vivo expansion of short-term HSPCs, but the effect on long-term repopulating hematopoietic stem cells (LTR-HSCs) remains uncertain. Here, we demonstrate that ex vivo culture of human adult HSPCs with DXI under low oxygen tension limits ER stress in LTR-HSCs and lineage-committed progenitors compared with normoxic cultures. A distinct HSC gene signature was upregulated in cells cultured with DXI in hypoxia and, after 21 days of culture, the frequency of LTR-HSCs increased 4.9-fold relative to uncultured cells and 4.2-fold compared with the normoxia + DXI group. NOTCH and hypoxia pathways intersected to maintain undifferentiated phenotypes in cultured HSPCs. Our work underscores the importance of mitigating ER stress perturbations to preserve functional LTR-HSCs in extended cultures and offers a clinically feasible platform for the expansion of human HSPCs.

Strongyloides stercoralis and HTLV-1 coinfection in CD34+ cord blood stem cell humanized mice: Alteration of cytokine responses and enhancement of larval growth.

Viral and parasitic coinfections are known to lead to both enhanced disease progression and altered disease states. HTLV-1 and Strongyloides stercoralis are co-endemic throughout much of their worldwide ranges resulting in a significant incidence of coinfection. Independently, HTLV-1 induces a Th1 response and S. stercoralis infection induces a Th2 response. However, coinfection with the two pathogens has been associated with the development of S. stercoralis hyperinfection and an alteration of the Th1/Th2 balance. In this study, a model of HTLV-1 and S. stercoralis coinfection in CD34+ umbilical cord blood hematopoietic stem cell engrafted humanized mice was established. An increased level of mortality was observed in the HTLV-1 and coinfected animals when compared to the S. stercoralis infected group. The mortality was not correlated with proviral loads or total viral RNA. Analysis of cytokine profiles showed a distinct shift towards Th1 responses in HTLV-1 infected animals, a shift towards Th2 cytokines in S. stercoralis infected animals and elevated TNF-α responses in coinfected animals. HTLV-1 infected and coinfection groups showed a significant, yet non-clonal expansion of the CD4+CD25+ T-cell population. Numbers of worms in the coinfection group did not differ from those of the S. stercoralis infected group and no autoinfective larvae were found. However, infective larvae recovered from the coinfection group showed an enhancement in growth, as was seen in mice with S. stercoralis hyperinfection caused by treatment with steroids. Humanized mice coinfected with S. stercoralis and HTLV-1 demonstrate features associated with human infection with these pathogens and provide a unique opportunity to study the interaction between these two infections in vivo in the context of human immune cells.

The Effect of Platelet Lysate on Expansion and Differentiation Megakaryocyte Progenitor Cells from Cord Blood CD34+ enriched Cells

Background: Umbilical cord blood hematopoietic stem cells (UCB-HSCs) are an attractive source for transplantation. The generation of megakaryocyte-committed cells could lead to shorten period of thrombocytopenia after HSCs transplantation. Platelet lysate (PL) unlike fetal bovine serum (FBS) can prevent immune problems as well as avert transmission of certain diseases to the recipient. In this study, the authors aimed to assess the effect of PL on UCB CD34+ cells expansion and megakaryocyte differentiation. Materials and Methods: In this experimental study, PL prepared and the subsequent isolation of UCB CD34+ cells were done by magnetic cell sorting. The isolated cells were cultivated in Iscove’s Modified Dulbecco’s medium (IMDM) supplemented with PL or FBS. Cell expansion was evaluated using Trypan blue. Furthermore, Flow cytometry using monoclonal antibodies (CD41-FITC and CD42b-PE) and the expression of specific genes including GATA1, GATA2, FLI1, NFE2, and RUNX1 via real-time PCR were performed to evaluate the megakaryocyte differentiation. Results: The results showed that PL insignificantly enhanced UCB CD34+ cell expansion (32.83± 8.47 fold in FBS and 41.67± 10.31 fold in PL containing media). Besides, flow cytometry results showed that expression of CD41 was increased markedly (37.81± 4.78 fold in FBS and 45.78 ± 7.37 in PL containing media, P-value &lt;0.05) but the elevation of CD42b (10.53 ± 2.13 and 13.20 ± 2.06 in FBS and PL containing media, respectively) was not significant (P-value = 0.051). The results of real-time PCR demonstrated a notable increase in GATA binding protein 1 (1.58, P-value &lt;0.01), GATA binding protein 2 (2.45, P-value &lt;0.001), RUNX family transcription factor 1 (1.60, P-value &lt;0.01), Fli-1 proto-oncogene (1.87, P-value &lt;0.001) in PL supplemented media, however, the increase of Nuclear Factor-Erythroid 2 gene expression was not significant in PL supplemented media (P-value = 0.11). Conclusion: PL improved UCB CD34+ cells expansion and megakaryocyte differentiation compared to FBS.

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.

Overcoming the UCB HSCs \u2013Derived NK cells Dysfunction through Harnessing RAS/MAPK, IGF-1R and TGF-\u03b2 Signaling Pathways

BackgroundThe natural killer (NK) cells differentiated from umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may be more suitable for cell-based immunotherapy compared to the NK cells from adult donors. This is due to the possibility to choose alloreactive donors and potentially more robust in vivo expansion. However, the cytotoxicity of UCB-HSC-derived NK cells against cancer cells might be suboptimal. To overcome this obstacle, we attempted to generate NK cells with potent antitumor activity by targeting RAS/MAPK, IGF-1R and TGF-β signaling pathways using IL-15, IGF-1 and SIS3 respectively.MethodsThe CD34 + cells were isolated from human UCB mononuclear cells through magnetic activation cell sorting (MACS) with purity of (≥ 90%) and were subjected to differentiate into NK cells. After 21 days of induction with SFTG36 (SCF, FLt-3L, TPO, GM-CSF, IL-3 and IL-6), IS721 (IGF-1, SIS3, IL-7 and IL-21) and IL-15/Hsp70 media, NK cells phenotypes were studied and their cytotoxicity against K562 human erythroleukemia cells and SKOV3 ovarian carcinoma cells was analyzed.ResultsThe NK cells induced in SFTG36/IS721 medium were selected for activation due to their higher expression of CD56 + 16 + CD3 − (93.23% ± 0.75) and mean fluorescence intensity (MFI) of NKG2D + (168.66 ± 20.00) and also a higher fold expansion potential (11.893 ± 1.712) compared to the other groups. These cells once activated with IL-15, demonstrated a higher cytotoxicity against K562 (≥ 90%; P ≤ 0.001) and SKOV3 tumor cells (≥ 65%; P ≤ 0.001) compared to IL-15/Hsp70-activated NK cells.ConclusionsThe differentiation of ex vivo expanded CD34 + cells through manipulation of RAS/MAPK, IGF-1R and TGF-β signaling pathways is an efficient approach for generating functional NK cells that can be used for cancer immunotherapy.