Human Umbilical Cord Blood CD34 Research Articles

Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the ex vivo expansion and maintenance of hematopoietic stem/progenitor cells

BackgroundMesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.MethodsFlow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.ResultsThe stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation.ConclusionsA novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell–cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.

Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

A Microwell Device for the Efficient Generation of Arrays of Microtissues and Humanized Bone Marrow Micro-Ossicles

(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation.

Human serum albumin promotes self-renewal and expansion of umbilical cord blood CD34+ hematopoietic stem/progenitor cells.

We investigated the effect of human serum albumin (HSA) on human umbilical cord blood (UCB) CD34+ hematopoietic stem/progenitor cells (HSPCs) cultured in vitro and transplanted in vivo. Human umbilical cord blood mononuclear cells were obtained by density gradient centrifugation. CD34+ cells were then sorted by CD34 conjugated magnetic microbeads. The sorted cells were cultured with or without HSA for 8 days in vitro. After 8 days, all cells were harvested for flow phenotyping and colony formation cell (CFC) experiments. The cells were injected into immunodeficient mice (NOD/Shi-scid/IL2Rγnull, NOG) via intravenous injections. From 4 weeks post-transplantation, flow cytometry was used to calculate human cell chimerism in the peripheral blood (PB) every 2 weeks. Flow phenotyping of human cell chimerism in bone marrow and spleen was calculated 16 weeks post-transplantation. Compared to the control group, CD34+ cells cultured with HSA increased significantly in vitro. The long-term engraftment of HSPCs and the hematopoietic multilineage reconstruction capacity were preserved by HSA. Normal engraftment of human cells could be maintained via HSA treatment could maintain normal engraftment of human cells in recipient PB. Here, we found that HSA was beneficial to maintaining CD34+ cell expansion and short-term colony formation in vitro and optimizing multilineage reconstitution in immunodeficient mice in vivo.

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.

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.

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.

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.

Biological Characteristics of Human Skeletal Muscle-Derived Pericytes/Perivascular Cells and Their Supporting Effects on Hematopoietic Stem Cell in Vitro

Mesenchymal stem/stromal cells (MSCs) as precursor cells of bone marrow stromal cells, in addition to the formation of the bone marrow microenvironment, secrete a variety of blood-related factors, promote hematopoietic recovery and reconstruction. However, mesenchymal stem/stromal cells are extremely rare within human bone marrow, which limit their experimental and clinical applications. Our previous study demonstrated that MSCs were derived from pericytes/perivascular cells (PCs), and PCs were the precursor of MSCs. PCs are widely distributed, especially in skeletal muscle tissue, which are rich in content, easy to be harvested with minimal damage, and rapid cell proliferation. However, very few researches have been done on whether human skeletal muscle-derived pericytes/perivascular cells (hMD-PCs) are involved in HSPCs expansion/proliferation, differentiation and possible hematopoietic regulation mechanisms. Based on the above research background and research ideas, in current study, we intend to isolate, purify and characterize the CD146+ hMD-PCs and further investigate their supporting effect on human umbilical cord blood CD34+ cells in vitro. Methods 1. hMD-PCs with phenotype CD146+CD56-CD34-CD144-CD45- were sorted from human skeletal muscle by enzymatic digestion and multiparameter fluorescence-activated cell sorting (MP-FACS), and their biological characteristic were conducted by detecting the surface marker of MSCs and inducing multilineage differentiation. 2. Human UCB CD34+cells were sorted by immunomagnetic beads, and then established the in vitro expansion culture system of UCB CD34+ cells co-culture with CD146+ hMD-PCs (experimental group), human bone marrow MSCs for feeder layer (positive control) and UCB CD34+ cells alone culture system (blank control). After 1 week, 2 weeks and 4 weeks of co-culture, the number of cells, the colony formation ability and immunophenotype were analysed. Result 1.CD146+ hMD-PCs were sorted by MP-FACS and the purity was (91.5±1.85)% (n=5); CD146+ hMD-PCs expressed mesenchymal surface markers CD73, CD90, CD105, CD44, and did not express hematopoietic cell and endothelial cell marker CD45, CD34, CD31. After induced culture, CD146+ hMD-PCs can differentiate into osteoblasts, chondrogenesis, adipocytes and myoblasts; 2.UCB CD34+ cells were sorted by magnetic beads which number was (9.18±3.50)×105, and then co-cultured with CD146+ hMD-PCs and human BM-MSCs at a density of 5×104/well, respectively. After 1, 2 and 4 weeks of co-culture, there were no statistically significant differences in cell number, colony formation ability and immunophenotype (CD45+cells, CD34+CD33-cells, CD14+cells, CD10+/CD19+cells) between CD146+ hMD-PCs for feeder layer culture system and BM-MSCs for feeder layer culture system (P>0.05, n=6). However, after 1 week, 2 weeks of co-culture, the both cells number of CD146+ hMD-PCs and human BM-MSCs culture system were significantly different from the blank control group, (P<0.01) and (P<0.001) respectively. The colony culture could not be performed due to low cell number of the blank control group. Conclusion CD146+ hMD-PCs, like human BM-MSCs, have hematopoietic support capacity in vitro; therefore CD146+ hMD-PCs can be used as another source of stromal cells for the expansion of hematopoietic stem cells. Disclosures No relevant conflicts of interest to declare.

Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture

AbstractHematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic “niche,” a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

Effects of human umbilical cord blood CD34+ cell transplantation in neonatal hypoxic-ischemia rat model

Perinatal brain injury can cause death in the neonatal period and lifelong neurodevelopmental deficits. Stem cell transplantation had been proved to be effective approach to ameliorate neurological deficits after brain damage. In this study we examine the effect of human umbilical cord blood CD34+ cells on model of neonatal rat hypoxic–ischemic brain damage and compared the neuroprotection of transplantation of CD34+ cells to mononuclear cells from which CD34+ cells isolated on neonatal hypoxic-ischemia rat model. Seven-day-old Sprague-Dawley rats were subjected to hypoxic-ischemic (HI) injury, CD34+ cells (1.5 × 104 cells) or mononuclear cells (1.0 × 106 cells) were transplanted into mice by tail vein on the 7 day after HI. The transplantation of CD34+ cells significantly improved motor function of rat, and reduced cerebral atrophy, inhibited the expression of glial fibrillary acidic protein (GFAP) and apoptosis-related genes: TNF-α, TNFR1, TNFR2, CD40, Fas, and decreased the activation of Nuclear factor kappa B (NF-κB) in damaged brain. CD34+ cells treatment increased the expression of DCX and lectin in ipsilateral brain. Moreover, the transplantation of CD34+ cells and MNCs which were obtained from the same amount of human umbilical cord blood had similar effects on HI. Our data demonstrated that transplantation of human umbilical cord blood CD34+ cells can ameliorate the neural functional defect and reduce apoptosis and promote nerve and vascular regeneration in rat brain after HI injury and the effects of transplantation of CD34+ cells were comparable to that of MNCs in neonatal hypoxic-ischemia rat model.

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

PRMT1-RBM15 axis regulates megakaryocytic differentiation of human umbilical cord blood CD34+ cells.

Protein arginine methyltransferase 1 (PRMT1) serves pivotal roles in various cellular processes. However, its role in megakaryocytic differentiation has not been clearly reported. The aim of the present study was to explore the role of the PRMT-RNA binding motif protein 15 (RBM15) axis in human MK differentiation and the feasibility of targeting PRMT1 for leukemia treatment. In the present study, PRMT1 was overexpressed and the RBM15 protein was knocked down in human umbilical cord blood cluster of differentiation (CD)34+ cells and the cells were then cultured in megakaryocytic differentiation medium. Flow cytometry was used to analyze CD41 and CD42 double-positive cells, as well as the protein expression levels of PRMT1 and RBM15. The results demonstrated that human cord blood CD34+ cells differentiate into mature MKs in high thrombopoitin medium, as demonstrated by CD41 and CD42 expression. Overexpression of PRMT1 in human umbilical cord blood CD34+ cells blocked the maturation of megakaryocytic cells. Knockdown of RBM15 by short hairpin RNA produced less mature MKs. PRMT1 inhibitor rescued PRMT1-blocked megakaryocytic differentiation. These results provide evidence for a novel role of PRMT1 in the negative regulation of megakaryocytic differentiation. PRMT1 may be a therapeutic target for leukemia treatment.

MicroRNA Microarray Profiling during Megakaryocyte Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells.

Objective In order to clarify the role of microRNAs (miRNA) in megakaryocyte differentiation, we ran a microRNA microarray experiment to measure the expression level of 961 human miRNA in megakaryocytes differentiated from human umbilical cord blood CD133+ cells. Materials and Methods In this experimental study, human CD133+ hematopoietic stem cells were collected from three human umbilical cord blood (UCB) samples, and then differentiated to the megakaryocytic lineage and characterized by flow cytometry, CFU-assay and ploidy analysis. Subsequently, microarray analysis was undertaken followed by quantitative polymerase chain reaction (qPCR) to validate differentially expressed miRNA identified in the microarray analysis. Results A total of 10 and 14 miRNAs were upregulated (e.g. miR-1246 and miR-148-a) and down-regulated (e.g. miR- 551b and miR-10a) respectively during megakaryocyte differentiation, all of which were confirmed by qPCR. Analysis of targets of these miRNA showed that the majority of targets are transcription factors involved in megakaryopoiesis. ConclusionWe conclude that miRNA play an important role in megakaryocyte differentiation and may be used as targets to change the rate of differentiation and further our understanding of the biology of megakaryocyte commitment.

HIF-1α-stabilizing agent FG-4497 rescues human CD34+ cell mobilization inresponse to G-CSF in immunodeficient mice.

Granulocyte colony-stimulating factor (G-CSF) is used routinely in the clinical setting to mobilize hematopoietic stem progenitor cells (HSPCs) into the patient's blood for collection and subsequent transplantation. However, a significant proportion of patients who have previously received chemotherapy or radiotherapy and require autologous HSPC transplantation cannot mobilize the minimal threshold of mobilized HSPCs to achieve rapid and successful hematopoietic reconstitution. Although several alternatives to the G-CSF regime have been tested, few are used in the clinical setting. We have shown previously in mice that administration of prolyl 4-hydroxylase domain enzyme (PHD) inhibitors, which stabilize hypoxia-inducible factor (HIF)-1α, synergize with G-CSF invivo to enhance mouse HSPC mobilization into blood, leading to enhanced engraftment via an HSPC-intrinsic mechanism. To evaluate whether PHD inhibitors could be used to enhance mobilization of human HSPCs, we humanized nonobese, diabetic severe combined immune-deficient Il2rg-/- mice by transplanting them with human umbilical cord blood CD34+ HSPCs and then treating them with G-CSF with and without co-administration of the PHD inhibitor FG-4497. We observed that combination treatment with G-CSF and FG-4497 resulted in significant mobilization of human lineage-negative (Lin-) CD34+ HSPCs and more primitive human Lin-CD34+CD38- HSPCs into blood and spleen, whereas mice treated with G-CSF alone did not mobilize human HSPCs significantly. These results suggest that the PHD inhibitor FG-4497 also increases human HSPC mobilization in a xenograft mouse model, suggesting the possibility of testing PHD inhibitors to boost HSPC mobilization in response to G-CSF in humans.

Evaluations of Intravenous Administration of CD34+ Human Umbilical Cord Blood Cells in a Mouse Model of Neonatal Hypoxic-Ischemic Encephalopathy

Several cell therapies have been explored as novel therapeutic strategies for neonatal encephalopathy because the benefits of current treatments are limited. We previously reported that intravenous administration of human umbilical cord blood (hUCB) CD34⁺ cells (hematopoietic stem cells/endothelial progenitor cells) at 48 h after insult exerts therapeutic effects in neonatal mice with stroke, i.e., permanent middle cerebral artery occlusion. Although neonatal stroke and hypoxic-ischemic encephalopathy (HIE) are grouped under the term “neonatal encephalopathy,” their pathogenesis differs. However, little is known about the differences in the effects of the same treatment between these 2 diseases. In this study, we investigated whether the same treatment protocol exerts therapeutic effects in neonatal mice with HIE. The treatment significantly ameliorated the decreased cerebral blood flow in the ischemic penumbra. Although the cylinder and rotarod tests showed a trend of amelioration of behavioral impairments from the treatment, these were not statistically significant. Morphological brain injuries were not altered by treatment. The cell administration did not cause any adverse effects apart from hyperactivity in the open-field test. Some of these findings are consistent with the results obtained in our previous study using a stroke model, but others are not. This study suggests that the treatment protocol needs to be optimized for each pathological condition.

Transplantation of human umbilical cord blood CD34+ cells into the liver of newborn NOD/SCID/IL-2Rγ null (NSG) mice after busulfan conditioning.

Transplantation of human umbilical cord blood CD34+ cells into the liver of newborn NOD/SCID/IL-2Rγ null (NSG) mice after busulfan conditioning.