Homeobox B4 Research Articles

Homeobox B4 optimizes the therapeutic effect of bone marrow mesenchymal stem cells on endotoxin-associated acute lung injury in rats

BackgroundAlveolar capillary endothelial cell (EC) injury has a pivotal role in driving acute respiratory distress syndrome (ARDS) progression and maintaining endothelial homeostasis. A previous ex vivo study revealed that overexpression of homeobox B4 (HOXB4) in bone marrow mesenchymal stem cells (BMSCs) enhanced protection against lipopolysaccharide (LPS)-induced EC injury by activating the Wnt/β-catenin pathway. This in vivo study was performed to verify whether BMSCs overexpressing HOXB4 exert similar protective effects on LPS-induced acute lung injury (ALI) in an animal model. MethodsThe ALI rat model was established by intraperitoneal injection of LPS. Wildtype BMSCs or BMSCs overexpressing HOXB4 were then injected via the tail vein. The lung characteristics of rats were visualized by computed tomography. Lung histopathological characteristics and collagen deposition were assessed by hematoxylin-eosin and Masson's staining, respectively, which were combined with the lung wet/dry ratio and proinflammatory factor levels in bronchoalveolar lavage fluid to further evaluate therapeutic effects. Expression of β-catenin and VE-cadherin was assessed by western blotting and immunofluorescence. ResultsCompared with wildtype BMSCs, overexpression of HOXB4 optimized the therapeutic effects of BMSCs, which manifested as improvements in lung exudation and histopathological features, reduced lung collagen deposition, amelioration of lung permeability, attenuation of lung inflammation, and enhanced expression of β-catenin and VE-cadherin proteins. ConclusionsHOXB4-overexpressing BMSCs optimized the protective effect against LPS-induced ALI by partially activating Wnt/β-catenin signaling.

Hsa-circ_0003420 induces apoptosis in acute myeloid leukemia stem cells and impairs stem cell properties

Objective To explore the effect of circular RNA-0003420 (circ_0003420) in acute myeloid leukemia (AML) relapse and leukemia stem cells (LSCs) properties. Materials and methods TRIzol reagent was used to extract total RNA from AML tissues or cells. Cell viability was assessed by Cell Counting Kit-8 and EdU staining assays. Cell apoptosis was determined by flow cytometry. Results Compared with normal hematopoietic stem cells, circular RNA hsa-circ_0003420 expression was considerably decreased in non-m3 AML stem cells. Furthermore, the lack of hsa-circ_0003420 is correlated with poor clinical results and impaired therapeutic effects in AML. Overexpression of hsa-circ_0003420 via transfection caused LSC death and inhibited the characteristics of leukemia tumor stem cells, including expression of Homeobox B4 (HOXB4), MYB, and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) axis. Furthermore, hsa-circ_0003420 targets the mRNA of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and hsa-circ_0003420 expression markedly repressed IGF2BP1 levels in LSCs. Restoration of IGF2BP1 eliminated the effect of hsa-circ_0003420 on the replication, apoptosis, and LSC phenotype of KG-1a cells. Discussion and conclusions Up-regulation of hsa-circ_0003420 expression in LSCs caused redox disorder, inflammation and apoptosis, suggesting that this protein could be used as a target for the treatment AML.

Overexpression of HOXB4 Promotes Protection of Bone Marrow Mesenchymal Stem Cells Against Lipopolysaccharide-Induced Acute Lung Injury Partially Through the Activation of Wnt/β-Catenin Signaling.

PurposePulmonary vascular endothelial cell (EC) injury is recognized as one of the pathological factors of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Bone marrow mesenchymal stem cell (BMSC)-based cytotherapy has attracted substantial attention over recent years as a promising therapeutic approach for ALI/ARDS; however, its use remains limited due to inconsistent efficacy. Currently, gene modification techniques are widely applied to MSCs. In the present study, we aimed to investigate the effect of BMSCs overexpressing Homeobox B4 (HOXB4) on lipopolysaccharide (LPS)-induced EC injury.MethodsWe used LPS to induce EC injury and established EC-BMSC coculture system using transwell chambers. The effect of BMSCs on ECs was explored by detecting EC proliferation, apoptosis, migration, tube formation, and permeability, and determining whether the Wnt/β-catenin pathway is involved in the regulatory mechanism using XAV-939, inhibitor of Wnt/ β-catenin.ResultsAs compared to BMSCWT, BMSCHOXB4 coculture promoted EC proliferation, migration, and tube formation after LPS stimulation and attenuated LPS-induced EC apoptosis and vascular permeability. Mechanistically, BMSCHOXB4 coculture prevented LPS-induced EC injury by activating the Wnt/β-catenin pathway, which is partially reversible by XAV-939. When cocultured with BMSCHOXB4, pro-inflammatory factors were dramatically decreased and anti-inflammatory factors were greatly increased in the EC medium compared to those in the LPS group (P<0.05). Additionally, when compared to BMSCWT coculture, the BMSCHOXB4 coculture showed an enhanced modulation of IL-6, TNF-α, and IL-10, but there was no statistically significant effect on IL-1β and IL-4.ConclusionCoculturing of BMSCHOXB4 prevented LPS-induced EC injury by reversing the inactivation of the Wnt/β-catenin signaling pathway. An in vivo study remains warranted to ascertain whether engraftment of BMSCHOXB4 can be an attractive strategy for the treatment of ALI/ARDS.

HOXB4 inhibits the proliferation and tumorigenesis of cervical cancer cells by downregulating the activity of Wnt/\u03b2-catenin signaling pathway

Homeobox B4 (HOXB4), which belongs to the homeobox (HOX) family, possesses transcription factor activity and has a crucial role in stem cell self-renewal and tumorigenesis. However, its biological function and exact mechanism in cervical cancer remain unknown. Here, we found that HOXB4 was markedly downregulated in cervical cancer. We demonstrated that HOXB4 obviously suppressed cervical cancer cell proliferation and tumorigenic potential in nude mice. Additionally, HOXB4-induced cell cycle arrest at the transition from the G0/G1 phase to the S phase. Conversely, loss of HOXB4 promoted cervical cancer cell growth both in vitro and in vivo. Bioinformatics analyses and mechanistic studies revealed that HOXB4 inhibited the activity of the Wnt/β-catenin signaling pathway by direct transcriptional repression of β-catenin. Furthermore, β-catenin re-expression rescued HOXB4-induced cervical cancer cell defects. Taken together, these findings suggested that HOXB4 directly transcriptional repressed β-catenin and subsequently inactivated the Wnt/β-catenin signaling pathway, leading to significant inhibition of cervical cancer cell growth and tumor formation.

Rapid and Efficient Conversion of Human Fibroblasts into Functional Neurons by Small Molecules.

SummaryRecent studies have demonstrated that human astrocytes and fibroblasts can be directly converted into functional neurons by small molecules. However, fibroblasts, as a potentially better cell resource for transplantation, are not as easy to reprogram as astrocytes regarding their fate to neurons, and chemically induced neurons (iNs) with low efficiency from fibroblasts resulted in limited application for the treatment of neurological disorders, including depression. Here, we report that human fibroblasts can be efficiently and directly reprogrammed into glutamatergic neuron-like cells by serially exposing cells to a combination of small molecules. These iNs displayed neuronal transcriptional networks, and also exhibited mature firing patterns and formed functional synapses. Importantly, iNs could integrate into local circuits after transplantation into postnatal mouse brain. Our study provides a rapid and efficient transgene-free approach for chemically generating neuron-like cells from human fibroblasts. Furthermore, our approach offers strategies for disease modeling and drug discovery in central nervous system disorders.

Homeobox B4 gene expression is upregulated by ghrelin through PI3-kinase signaling pathway in rat's bone marrow stromal cells.

Ghrelin, a 28 amino acid peptide, has diverse physiological roles. Phosphatidylino-sitol-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) are involved in some of the recognized actions of ghrelin. It has been shown that ghrelin upregulates HOXB4 gene expression but the real mechanism of this effect is not clear. Rat bone marrow stromal cells (BMSCs) were cultured in DMEM. BMSCs were treated with ghrelin (100 μM) for 48 h. Real-time PCR for HOXB4 was performed from Control (untreated BMSCs), BG (BMSCs treated with 100 µM ghrelin), PD (BMSCs treated with 10 µM PD98059, a potent inhibitor of mitogen-activated protein kinase, and 100 µM ghrelin), LY (BM-SCs treated with 10 µM LY294002, a strong inhibitor of phosphoinositide 3-kinase, and 100 µM ghrelin) and SY (BMSCs treated with 10 µM LY294002 plus 10 µM PD98059, and 100 µM ghrelin) groups. Relative gene expression changes were determined using Relative expression software tool 9 (REST 9). HOXB4 gene has been overexpressed in ghrelin-treated BMSCs (p<0.05). PI3K inhi-bition by LY294002 significantly downregulated the ghrelin-induced overexpression of HOXB4 (p<0.05). We can conclude that ghrelin, through PI3K/Akt pathway, may improve BMSC transplantation potency by reducing its apoptosis. Moreover, upregulating HOXB4 in BMSC and its possible differentiation to HSCs might in the future open the doors to new treatment for hematologic disorders. Therefore, activating the PI3K/Akt pathway, instead of using a non-specific inducer, could be the principal point to increase the efficiency of BMSC-based cell therapies in the future.

HOXB4 knockdown enhances the cytotoxic effect of paclitaxel and cisplatin by downregulating ABC transporters in ovarian cancer cells

Therapeutic effects of anti-cancer drugs for ovarian cancer were limited due to the rapid development of chemotherapy resistance. The aim of this study was to test whether knockdown of Homeobox B4 (HOXB4) enhanced the cytotoxic effect of paclitaxel and cisplatin in ovarian cancer cells. HOXB4 expressions at mRNA and protein levels were upregulated in Taxol-resistant A2780 (A2780/Taxol) and DDP-resistant SKOV-3 (SKOV-3/DDP) cells. HOXB4 knockdown enhanced the cytotoxic effects of Taxol and DDP in A2780/Taxol and SKOV-3/DDP cells, respectively. HOXB4 silencing suppressed the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and reduced the expression of ABCB1, ABCC1 and ABCG2 in ovarian cancer cells. PI3K inhibitor LY294002 or siRNA targeting Akt (si-Akt) treatment inhibited cell viability, decreased protein levels of ABCB1, ABCC1 and ABCG2, and increased LDH release in A2780/Taxol and SKOV-3/DDP cells. These findings revealed that HOXB4 knockdown enhanced the cytotoxic effects of Taxol and DDP by downregulating ABC transporters via inhibiting the PI3K/Akt pathway in ovarian cancer cells.

Homeobox B4 inhibits breast cancer cell migration by directly binding to StAR-related lipid transfer domain protein 13.

The present study aimed to investigate the role of homeobox B4 (HOXB4) in breast cancer. Analysis of The Cancer Genome Atlas data revealed that HOXB4 expression was positively associated with expression of the StAR-related lipid transfer domain protein 13 (STARD13), and the overall survival of patients with breast cancer. Immunohistochemistry and quantitative polymerase chain reaction assays demonstrated that HOXB4 expression was downregulated in breast cancer tissues compared with adjacent normal tissues and was additionally positively associated with STARD13 expression. HOXB4 promoted STARD13 expression in breast cancer cells. Chromatin immunoprecipitation and luciferase reporter assays confirmed that HOXB4 directly bound to the STARD13 promoter. Additionally, HOXB4 inhibited breast cancer cell migration and the epithelial-mesenchymal transition through the STARD13/Ras homolog (Rho) family member A/Rho associated protein kinase signaling pathway. HOXB4 overexpression enhanced the sensitivity of breast cancer cells to doxorubicin and reversed resistance in doxorubicin-resistant cells. Overall, the results indicated that HOXB4 inhibited breast cancer cell migration and enhanced the sensitivity of breast cancer cells to doxorubicin by targeting STARD13.

HOXB4 knockdown reverses multidrug resistance of human myelogenous leukemia K562/ADM cells by downregulating P-gp, MRP1 and BCRP expression via PI3K/Akt signaling pathway.

Multidrug resistance (MDR) plays a pivotal role in human chronic myelogenous leukemia (CML) chemotherapy failure. MDR is mainly associated with the overexpression of drug efflux transporters of the ATP-binding cassette (ABC) proteins. Phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with multidrug resistance1 (MDR1)/P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) expression in many human malignancies. Homeobox (HOX) B4, a member of the HOX gene family, has been reported to be correlated with occurrence, development, poor prognosis and drug resistance of human leukemia. In the present study, HOXB4 expression was analyzed in K562 cell line and its MDR subline K562/ADM. Compared with K562 cells, drug-resistant K562/ADM cells demonstrated evidently higher HOXB4 expression. In addition, we firstly investigated the reversal effect of HOXB4 deletion on K562/ADM cells and the underlying mechanism. The Cell Counting kit-8 (CCK-8) and flow cytometry assays showed that knockdown of HOXB4 enhanced chemosensitivity and decreased drug efflux in K562/ADM cells. Moreover, HOXB4 knockout led to downregulation of P-gp, MRP1 and BCRP expression and PI3K/Akt signaling activity, suggesting that repression of HOXB4 might be a key point to reverse MDR of K562/ADM cells.

No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model

Umbilical cord blood transplant continues to increase in prevalence as a treatment option for various hematopoietic and immune disorders. Because of the limited number of cells available in a single cord blood unit, investigators have explored methods of increasing cell dose before transplant, including overexpression of the homeobox B4 (HOXB4) transcription factor. We have previously reported the development of leukemia in several nonhuman primate (NHP) subjects transplanted with HOXB4-expanded bone marrow cells at approximately 2 years posttransplant. Here, we provide long-term data for a NHP receiving a HOXB4-expanded cord blood graft. Longitudinal follow-up included gene marking analysis, complete blood counts, morphologic/pathologic assessment, phenotypic analysis of subsets, and retroviral integration site analysis. In each of these independent assays, we saw no indication of clonal dominance, and all signs pointed toward normal, healthy hematopoiesis. Furthermore, in-depth clonal analysis of an animal that developed leukemia after transplantation of HOXB4-modified bone marrow cells showed that dominant clones could be detected as early as 6 months posttransplant using the genomic analysis technique detailed here. Parallel analysis of the cord blood transplant macaque showed no such sites. These findings demonstrate the ability to study the use of gene-modified and expanded cord blood cells in a NHP model.

Inability of HOXB4 to enhance self-renewal of malignant B cells: Favorable profile for the expansion of autologous hematopoietic stem cells

Leukemic stem cells share self-renewal properties and slow proliferation with hematopoietic stem cells. Based on expression signatures, it has been suggested that these cells use the same molecular pathways for these processes. However, it is not clear whether leukemic stem cells also respond to factors known to enhance the self-renewal activity of hematopoietic stem cells. The transcription factor homeobox B4 (HOXB4) is known to induce expansion of mouse hematopoietic stem cells. The recombinant TAT-HOXB4 protein also expands human CD34+ cells. In this study we investigated whether overexpression of HOXB4 could increase leukemic initiating cell numbers, an issue that is crucial to its clinical usage. A transgenic mouse model for E2A-PBX1 induced pre-B acute lymphoblastic leukemia was used in combination with HOXB4 transgenic mice to test oncogenic interactions between HOXB4 and E2A-PBX1. The frequency of leukemic initiating cells retrovirally overexpressing HOXB4 was measured by transplantation at limiting dilution and evaluation of leukemia development in recipient mice. Moreover, human B cell lines were evaluated for their colony forming cell potential upon exposure to TAT-HOXB4 protein. Our data with the mouse models show that HOXB4 neither accelerates the generation of E2A-PBX1 B cell leukemia nor expands the number of leukemia initiating cells. Additionally, the growth or colony forming cell proportions of human B cell lines was not changed by HOXB4, suggesting that human B leukemic initiating cells are not affected by HOXB4.

Long-term follow-up study on the engraftment of human hematopoietic stem cells in sheep.

Xenograft models of human hematopoiesis are essential to the study of the engraftment and proliferative potential of human hematopoietic stem cells (HSCs) in vivo. Immunodeficient mice and fetal sheep are often used as xenogeneic recipients because they are immunologically naive. In this study, we transplanted human HSCs into fetal sheep and assessed the long-term engraftment of transplanted human HSCs after birth. Fourteen sheep were used in this study. In 4 fetal sheep, HSCs were transduced with homeo-box B4 (HOXB4) gene before transplantation, which promoted the expansion of HSCs. Another 4 fetal sheep were subjected to non-myeloablative conditioning with busulfan. Seven of these 8 sheep showed successful engraftment of human HSCs (1–3% of colony-forming units) as assessed after the birth of fetal sheep (5 months post-transplantation), although HOXB4-transduced HSCs showed sustained engraftment for up to 40 months. Intact HSCs were transplanted into six non-conditioned fetal sheep, and human colony-forming units were not detected in the sheep after birth. These results suggest that, as compared with mouse models, where the short lifespan of mice limits long-term follow-up of HSC engraftment, the fetal sheep model provides a unique perspective for evaluating long-term engraftment and proliferation of human HSCs.

Studies on HOXB4 Expression During Differentiation of Human Cytomegalovirus-infected Hematopoietic Stem Cells into Lymphocyte and Erythrocyte Progenitor Cells

We investigated the role of homeobox B4 (HOXB4) mRNA/protein expression induced by human cytomegalovirus (HCMV) and/or all-trans retinoic acid (ATRA) in proliferation and committed differentiation of human cord blood hematopoietic stem cells (HSCs) into colony-forming-units of T-lymphocyte (CFU-TL) and erythroid (CFU-E) progenitors in vitro. Twelve cord blood samples were collected from the fetal placenta umbilical vein and cultured in vitro. The proliferation and differentiation of cord blood HSCs into CFU-TL and CFU-E were continuously disrupted with HCMV-AD169 and/or 6 × 10(-8) mol/l of ATRA. HOXB4 mRNA/protein expression in CFU-TL and CFU-E was detected in control, ATRA, HCMV and ATRA + HCMV groups on days 3, 7, and 12 of culture by fluorescent qRT-PCR/western blot. We found that HOXB4 mRNA/protein expression was detectable on day 3, increased on day 7 and was highest on day 12. HOXB4 mRNA/protein expression in HCMV group was downregulated compared with control group (P < 0.05). However, the levels were significantly upregulated in HCMV + ATRA group compared with HCMV group (P < 0.05). We concluded that the abnormal HOXB4 mRNA/protein expression induced by HCMV could play a role in hematopoietic damage. ATRA, at the concentration used, significantly up-regulated HOXB4 mRNA/protein expression in normal lymphocyte and erythrocyte progenitor cells as well as in HCMV-infected cells.

The Role of rap1b in Hoxb4 Transgenic Zebrafish Line,

Abstract 3413Self-renewal – the generation of daughter cells having the same regenerative properties as the parental cell – is the defining feature of hematopoietic stem cells (HSCs). Homeobox b4 (Hoxb4) is the key gene shown capable of expanding hematopoietic stem cells (HSCs) in vivo and in vitro. It is imperative to establish a stable Hoxb4 overexpressing transgenic live animal model. Here, using a Cre-loxP system, we established a transgenic zebrafish line that stably overexpressing enhanced green fluorescent protein (EGFP) – tagged hoxb4 protein under the control of hemangioblast-specific lmo2 promoter. The results indicate that Hoxb4 favors hematopoietic stem cell development of hemangioblasts in vivo. The mechanisms of Hoxb4-mediated HSC proliferation and the reason why Hoxb4 overexpression can cause retardation of specific and terminal blood cells in vivo are still unknown.Rap1 belongs to the Ras subfamily of small GTP-binding proteins. Rap1 can regulate cell proliferation, differentiation, and adhesion through distinct mechanisms. So we want to know whether Hoxb4 overexpression can influence Rap1b expression. The expression difference of Rap1b in blank control group(Tubegin Wild type), experimental control group (Tg(zLmo2:LDL-EGFP)×Tg(zLmo2:Cre)), experimental group (Tg(zLmo2:LDL-Hoxb4-EGFP) ×Tg(zLmo2:Cre)) zebrafish embryo were detected by whole embryo in situ hybridization (WISH) with Rap1b antisense mRNA probe. It was found that Rap1b expression was extended from 30 to 36 hour postfertilization(hpf) when Hoxb4 was overexpressed. In the Hoxb4 expressed lines, rap1b was upregulated and strongly expressed in the caudal hematopoietic tissue (CHT) at 36 hpf. Then, the EGFP labeled hematopoietic cell in transgenic line were collected with the fluorescence activated cell sorter (FACS). The Rap1b mRNA expression of sorting cell were compared by SqRT-PCR (Semi-quantitative Reverse Transcription and Polymerase Chain Reaction). The gene expressions of Rap1b was upregulated in Hoxb4 experimental group. These results indicate that Rap1b gene maybe is downstream target gene of Hoxb4 gene in hematopoietic system of zebrafish. Disclosures:No relevant conflicts of interest to declare.

Combination of HOXB4 and Delta-1 ligand improves expansion of cord blood cells

AbstractUmbilical cord blood (UCB) is an attractive cell source for hematopoietic cell transplantation (HCT). Here we examine whether the combination of homeobox B4 (HOXB4) and Delta-1 ligand (DL) synergize when used together. Monkey and human UCB CD34+ cells were transduced with a HOXB4-expressing gammaretroviral vector and cultured with DL. Individual and combined effects of HOXB4 and DL were assessed by colony-forming unit assays, flow cytometry, and nonobese diabetic/severe combined immune deficienct mouse transplantation. The presence of DL yielded higher percentage of CD34+ and CD7+ cells and lower percentages of CD14+ cells than non-DL cultures. Furthermore, HOXB4 yielded higher percentages of CD34+ and CD14+ cells than non-HOXB4 cultures. Interestingly, coculture with DL-expressing OP9 cells resulted in better maintenance of HOXB4 than culture in DL-conditioned medium. Culture of HOXB4-transduced human cells in the presence of DL yielded enhanced generation of repopulating cells with higher levels of engraftment of human CD45+, CD34+, CD3+, CD20+, and CD41+ cells compared with either factor individually. Our results demonstrate enhanced generation of hematopoietic progenitors by combining HOXB4 and DL; addition of DL further enhances expansion of multipotent cells capable of repopulating lymphoid and megakaryocyte lineages, which is not observed with HOXB4 alone.

Ex vivo expansion of human HSCs with Sendai virus vector expressing HoxB4 assessed by sheep in utero transplantation

The homeobox B4 (HoxB4) gene promotes expansion of hematopoietic stem cells (HSCs). However, frequent development of leukemia in large animals due to retrovirally transduced HoxB4 gene has been reported. To prevent tumorigenesis, we developed a nonintegrating and nonreplicating Sendai virus vector that did not contain the phosphoprotein gene (SeV/ΔP), which enabled clearance of the vector and transgene shortly after transduction. We tested the SeV/ΔP vector expressing the HoxB4 gene (SeV/ΔP/HoxB4) for the ex vivo expansion of human cord blood CD34(+) cells (HSCs) using a sheep in utero transplantation assay. Human HSCs were ex vivo-expanded by transduction with SeV/ΔP/HoxB4 vector and transplanted into the abdominal cavity of fetal sheep. The engraftment of human HSCs in the lambs was quantitatively evaluated by hematopoietic colony-forming unit assays. After transplantation, the HoxB4-transduced HSCs contributed to longer-period (up to 20 months) repopulation in sheep, and human hematopoietic progenitors were detected more frequently in the bone marrow of the HoxB4 group as compared with the control untreated group (p < 0.05). The expansion of human HSCs with the SeV/ΔP/HoxB4 vector was comparable with previously reported retroviral vectors expressing HoxB4. The SeV/ΔP/HoxB4 vector and the transgene were cleared from the recipient sheep and leukemia was not detected at 20 months post-transplantation. The SeV/ΔP vector would be suitable for transient expression of HoxB4 in human CD34(+) cells. In addition, the SeV/ΔP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors.

Successful Treatment of Metachromatic Leukodystrophy Using Bone Marrow Transplantation of HoxB4 Overexpressing Cells

To evaluate the contribution of bone marrow (BM) cells to treat neurological disorders, we examined the effectiveness of BM cells expressing the homeobox B4 (HoxB4) gene to cure mice with metachromatic leukodystrophy (MLD) through transplantation. Increased number of donor cells was observed in brains of the MLD mice transplanted with HoxB4-transduced BM cells (B4MLD) in contrast to those transplanted with control green fluorescent protein (GFP)-transduced BM cells (MIGMLD). Immunohistochemical staining showed that most of the GFP(+) cells were Iba1(+) microglia. In addition, O4(+) oligodendrocytes were identified only in the B4MLD brains but not in the MIGMLD brain. Alcian blue staining showed that accumulation of sulfatide was dramatically reduced in brain tissue from B4MLD mice, and there was a corresponding improvement in the animals' ability to walk a balance beam 8 months after transplantation. Thus transplantation of BM cells overexpressing HoxB4 appears to effectively prevent the progression of MLD in this mouse model. These findings support the idea that hematopoietic stem cells (HSCs) transduced with a HoxB4 expression vector could be the useful carriers of therapeutic proteins into the brain for regeneration of oligodendrocytes to treat such demyelinating disorders as MLD, Krabbe disease, and multiple sclerosis.

Suppression of embryonic lung branching morphogenesis by antisense oligonucleotides against HOM/C homeobox factors

The role of HOM/C homeobox genes on rat embryonic lung branching morphogenesis was investigated using the lung bud explant culture system in an air/liquid interface. Knock down of homeobox b3 and b4 expression by antisense oligonucleotide treatment repressed airway branch formation, while antisense oligonucleotide against homeobox a3 showed no effect. Addition of antisense Hoxb3 oligonucleotide resulted in upregulation of collagen type III mRNA and fibroblast growth factor 10 mRNA, while that of the T-box regulatory factor-4 was decreased. Consequently, expression of Clara cell-specific secretory protein was decreased. These results suggest a critical role for homeobox b3 and b4 genes in lung airway branching morphogenesis.

Purified Recombinant TAT-Homeobox B4 Expands CD34+Umbilical Cord Blood and Peripheral Blood Progenitor CellsEx Vivo

Hematopoietic stem cells (HSCs) are commonly used in clinical transplantation protocols to treat a variety of diseases. However, efficient transplantation requires a substantial amount of HSCs from different sources and may require expansion. Therefore, effective expansion of HSCs remains a technical hurdle blocking the development of advanced cell therapies. The product of the human homeobox B4 (HOXB4) gene was recently demonstrated to effectively expand HSCs from umbilical cord blood (UCB) or bone marrow in either a retroviral or recombinant protein form. Our study purified TAT-HOXB4 proteins and demonstrated their ability to expand UCB and peripheral blood (PB) progenitor cells. The results showed that the TAT-HOXB4 gene product expanded the CD34(+) progenitor cells from UCB and PB by approximately 7.5-fold. The results from a semisolid cloning assay, a human long-term culture-initiating cell assay, and a nonobese diabetic-severe combined immunodeficiency mice repopulating assay showed that TAT-HOXB4 expanded hematopoietic progenitor cells while retaining their repopulating capacity and multipotency. TAT-HOXB4 protein also expanded engrafted stem cells that were previously expanded in a secondary transplantation assay. The results demonstrated the feasibility of using TAT-HOXB4 to expand UCB and PB progenitor cells, which are readily available to treat different hematological malignancies and nonhematological diseases.

Safe and Efficient Expansion of Human HSC with Sendai Virus Vector Expressing HoxB4 in Fetal Sheep.

Abstract 693 Background:Homeobox B4 (HoxB4) has been shown to be a potent stem cell self-renewal gene, especially in hematopoietic stem cells (HSC). Accumulating evidence from murine studies indicates that the overexpression of HoxB4 enhances in vivo and ex vivo expansion of HSC. Although no leukemia has been observed after transplantation of HoxB4-transduced cells in murine models, the study using large animals such as dogs and non-human primates with retroviral vectors expressing HoxB4 showed the frequent development of leukemia. Regarding retroviral vectors expressing HoxB4, there is another concern, that is, insertional leukemogenesis, which has been elucidated in the hematopoietic stem cell gene therapy for X-SCID. To avoid the insertional mutagenesis, other vectors may be considered, including Epstein-Barr nuclear antigen (EBNA)-1 based episomal vectors or the transposon; however, problems are left, i.e. low transduction efficiency with EBNA vectors and unclear safety with transposon vectors. To avoid both the persistent HoxB4 expression and insertional mutagenesis leading to leukemogenesis, we have developed a new type of Sendai virus (SeV) vector; it lacks the polymerase gene, namely P-defective SeV (SeV/δP) vector. SeV is an enveloped virus with a non-segmented, negative-stranded RNA genome. SeV-based vectors are non-integrating, cytoplasmic vectors. They replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into chromosomal DNA of the host. We have previously shown that the transduction efficiency of human CD34+ cells with the SeV vector was very high; around 70% (100 multiplicity of infections). On the other hand, SeV/δP vectors are incapable of self-replication, thus enabling transient gene expression without spoiling their ability to efficiently transduce CD34+ cells. Here, using the SeV/δP vector expressing HoxB4 (SeV/δP/HoxB4 vector), we examined the effectiveness and safety of human HSC expansion after in utero transplantation to fetal sheep. Methods:After enrichment of CD34+ cells from cryopreserved human umbilical cord blood, these cells were repeatedly exposed to SeV/δP/HoxB4 vector every 24 h for 4 days. The transduced cells (3.2–11.7 × 105) were transplanted into the abdominal cavity of fetal sheep at 45–50 gestational days (full term, 147 days) that have premature immune system (HoxB4 group, n = 4; control group, n = 4). The engraftment of hematopoietic cells derived from human HSC in the lambs after birth was quantitatively evaluated by colony PCR of the bone marrow. The development of leukemia was assessed by regular sampling of peripheral blood and bone marrow. Results:The human–sheep chimeric ratio in the bone marrow of HoxB4 group was calculated 4.8-times higher than that of control group after birth, as assessed by colony PCR. The SeV genome was no longer detectable in the bone marrow and peripheral blood of lambs as assessed by RNA-PCR, confirming the SeV vectors were cleared. No leukemia developed in any of the sheep in either group at present (at 12 months after transplantation). Conclusion:The SeV/δP vector would be suitable for transient expression of HoxB4 in human CD34+ cells, enabling 4.8-times expansion of human HSC as assessed by their repopulating ability in sheep. The expansion of human HSC with the SeV vector was comparable to that with HoxB4-retroviral vectors. In addition, the SeV/δP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors. Disclosures:No relevant conflicts of interest to declare.