CAR-NK cells from engineered pluripotent stem cells: Off-the-shelf therapeutics for all patients.

Clinical success of adoptive cell therapy with chimeric antigen receptor (CAR) T cells for treating hematological malignancies has revolutionized the field of cellular immunotherapy. However, due to the nature of utilizing autologous T cells, affordability and availability are major hurdles, in addition to scientific challenges relating to CAR-T therapy optimization. Natural killer (NK) cell is a specialized immune effector cell type that recognizes and kills targets without human leukocyte antigen (HLA) restriction and prior sensitization. CAR-NK cells do not cause graft vs host disease and can be obtained from unrelated donors as well as pluripotent stem cells (PSC), representing an ideal off-the-shelf therapeutics readily available for patients. Furthermore, unlike cytotoxic T cells, NK cells specifically target and eliminate cancer stem cells, which are the cells causing relapse and metastasis. PSCs can be genetically manipulated and engineered with CARs at the pluripotent stage, which allows the establishment of permanent, stable, and clonal PSC-CAR lines for the manufacture of unlimited homogenous CAR-NK cells. Multiple master PSC-CAR cell banks targeting a variety of antigens for cancer, viral infection, and autoimmune diseases provide inexhaustible cell sources for all patients. Development of a next-generation 3D bioreactor platform for PSC expansion and NK cell production overcomes major barriers related to cost and scalability for CAR-NK product.

Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

OBJECTIVE: To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. METHODS: Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. RESULTS: In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 µl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 µl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. CONCLUSION: By integrating into damaged retinal vessels and differentiating into ECs, intravitreally administered hESC-HBs may have partially repaired a retinal vascular injury caused by OIR model and DR.

Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.

Transdifferentiation of Human Hair Follicle Mesenchymal Stem Cells into Red Blood Cells by OCT4.

Shortage of red blood cells (RBCs, erythrocytes) can have potentially life-threatening consequences for rare or unusual blood type patients with massive blood loss resulting from various conditions. Erythrocytes have been derived from human pluripotent stem cells (PSCs), but the risk of potential tumorigenicity cannot be ignored, and a majority of these cells produced from PSCs express embryonic ε- and fetal γ-globins with little or no adult ß-globin and remain nucleated. Here we report a method to generate erythrocytes from human hair follicle mesenchymal stem cells (hHFMSCs) by enforcing OCT4 gene expression and cytokine stimulation. Cells generated from hHFMSCs expressed mainly the adult ß-globin chain with minimum level of the fetal γ-globin chain. Furthermore, these cells also underwent multiple maturation events and formed enucleated erythrocytes with a biconcave disc shape. Gene expression analyses showed that OCT4 regulated the expression of genes associated with both pluripotency and erythroid development during hHFMSC transdifferentiation toward erythroid cells. These findings show that mature erythrocytes can be generated from adult somatic cells, which may serve as an alternative source of RBCs for potential autologous transfusion.

Scalable generation of universal platelets from human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) provide a potentially replenishable source for the production of transfusable platelets. Here, we describe a method to generate megakaryocytes (MKs) and functional platelets from iPSCs in a scalable manner under serum/feeder-free conditions. The method also permits the cryopreservation of MK progenitors, enabling a rapid "surge" capacity when large numbers of platelets are needed. Ultrastructural/morphological analyses show no major differences between iPSC platelets and human blood platelets. iPSC platelets form aggregates, lamellipodia, and filopodia after activation and circulate in macrophage-depleted animals and incorporate into developing mouse thrombi in a manner identical to human platelets. By knocking out the ß2-microglobulin gene, we have generated platelets that are negative for the major histocompatibility antigens. The scalable generation of HLA-ABC-negative platelets from a renewable cell source represents an important step toward generating universal platelets for transfusion as well as a potential strategy for the management of platelet refractoriness.

Human ESC-derived MSCs outperform bone marrow MSCs in the treatment of an EAE model of multiple sclerosis.

Current therapies for multiple sclerosis (MS) are largely palliative, not curative. Mesenchymal stem cells (MSCs) harbor regenerative and immunosuppressive functions, indicating a potential therapy for MS, yet the variability and low potency of MSCs from adult sources hinder their therapeutic potential. MSCs derived from human embryonic stem cells (hES-MSCs) may be better suited for clinical treatment of MS because of their unlimited and stable supply. Here, we show that hES-MSCs significantly reduce clinical symptoms and prevent neuronal demyelination in a mouse experimental autoimmune encephalitis (EAE) model of MS, and that the EAE disease-modifying effect of hES-MSCs is significantly greater than that of human bone-marrow-derived MSCs (BM-MSCs). Our evidence also suggests that increased IL-6 expression by BM-MSCs contributes to the reduced anti-EAE therapeutic activity of these cells. A distinct ability to extravasate and migrate into inflamed CNS tissues may also be associated with the robust therapeutic effects of hES-MSCs on EAE.

3D microcarrier system for efficient differentiation of human pluripotent stem cells into hematopoietic cells without feeders and serum [corrected].

BACKGROUND: Human embryonic stem cells (hESCs) have been derived and maintained on mouse embryonic fibroblast feeders to keep their undifferentiated status. To realize their clinical potential, a feeder-free and scalable system for large scale production of hESCs and their differentiated derivatives is required. MATERIALS & METHODS: hESCs were cultured and passaged on serum/feeder-free 3D microcarriers for five passages. For embryoid body (EB) formation and hemangioblast differentiation, the medium for 3D microcarriers was directly switched to EB medium. RESULTS: hESCs on 3D microcarriers maintained pluripotency and formed EBs, which were ten-times more efficient than hESCs cultured under 2D feeder-free conditions (0.11 ± 0.03 EB cells/hESC input 2D vs 1.19 ± 0.32 EB cells/hESC input 3D). After replating, EB cells from 3D culture readily developed into hemangioblasts with the potential to differentiate into hematopoietic and endothelial cells. Furthermore, this 3D system can also be adapted to human induced pluripotent stem cells, which generate functional hemangioblasts with high efficiency. CONCLUSION: This 3D serum- and stromal-free microcarrier system is important for future clinical applications, with the potential of developing to a GMP-compatible scalable system.

Potential clinical applications for human pluripotent stem cell-derived blood components.

The ability of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) to divide indefinitely without losing pluripotency and to theoretically differentiate into any cell type in the body makes them highly attractive cell sources for large scale regenerative medicine purposes. The current use of adult stem cell-derived products in hematologic intervention sets an important precedent and provides a guide for developing hESC/iPSC based therapies for the blood system. In this review, we highlight biological functions of mature cells of the blood, clinical conditions requiring the transfusion or stimulation of these cells, and the potential for hESC/iPSC-derivatives to serve as functional replacements. Many researchers have already been able to differentiate hESCs and/or iPSCs into specific mature blood cell types. For example, hESC-derived red blood cells and platelets are functional in tasks such as oxygen delivery and blood clotting, respectively and may be able to serve as substitutes for their donor-derived counterparts in emergencies. hESC-derived dendritic cells are functional in antigen-presentation and may be used as off-the-shelf vaccine therapies to stimulate antigen-specific immune responses against cancer cells. However, in vitro differentiation systems used to generate these cells will need further optimization before hESC/iPSC-derived blood components can be used clinically.

alpha-Thalassemia-like globin gene expression by primitive erythrocytes derived from human embryonic stem cells.

Under culture conditions that promote hematopoietic differentiation, human embryonic stem cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of early-stage human embryos. The globin chain distribution of these cells is similar to that seen during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of gamma-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly unbalanced, with a substantial decrease of alpha-like globin chain synthesis in relation to that of their beta-like globins, a pattern characteristically associated with alpha-thalassemia (alpha-thal). This pattern of unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize predominantly embryonic-stage globins.

Directed differentiation of red blood cells from human embryonic stem cells.

Human embryonic stem cells (hESC) represent a new source of stem cells that can be propagated and expanded in vitro indefinitely, providing a potentially inexhaustible and donorless source of cells for human therapy. The ability to create banks of hESC lines with matched or reduced incompatibility could potentially reduce or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols altogether, for example, O-type RhD(-) lines for generation of universal red blood cells (RBC). Hematopoietic differentiation of hESCs has been extensively investigated in vitro, and hematopoietic precursors as well as differentiated progeny representing erythroid, myeloid, macrophage, megakaryocytic, and lymphoid lineages have been identified in differentiating hESC cultures. Previous studies also generated primitive erythroid cells from hESCs by embryoid body (EB) formation and coculturing with stromal cells. However, the efficient and controlled differentiation of hESCs into homogeneous RBC populations with oxygen-carrying capacity has not been previously achieved. In this chapter, we describe a robust system that can efficiently generate large numbers of hemangioblasts from multiple hESC lines using well-defined conditions and produce functional homogeneous RBCs with oxygen-carrying capacity in large scale. The homogeneous erythroid cells can be used for further mechanism studies.

Hemangioblastic derivatives from human induced pluripotent stem cells exhibit limited expansion and early senescence.

Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods.

Therapeutic angiogenesis in diabetic apolipoprotein E-deficient mice using bone marrow cells, functional hemangioblasts and metabolic intervention.

OBJECTIVE: Peripheral arterial disease (PAD) is a major health problem especially when associated to concomitant diabetes and hypercholesterolemia. Hyperglycemia with an overwhelming generation of oxygen radicals and formation of glycation end-products exacerbates oxidation-sensitive mechanisms activated by tissue ischemia. Administration of autologous bone marrow cells (BMC) is an increasing notable intervention to induce therapeutic angiogenesis, ameliorated by metabolic intervention (MT). Recently, hemangioblasts (HS) with functional properties were isolated. METHODS: The effects of integrate regimen with intravenous BMC, HS, and MT (1.0% vitamin E, 0.05% vitamin C, and 6% l-arginine) were examined in the ischemic hindlimb of ApoE(-/-) diabetic and non-diabetic. Blood flow ratio was monitored by use of a laser Doppler blood flowmeter. Capillary density was determined in sections of the adductor and semimembranous muscles with antibody against CD31. RESULTS: BMC or HS alone, and BMC plus HS increased blood flow and capillary densities and decreased interstitial fibrosis. These effects were amplified by additional MT, at least in part, through the nitric oxide pathway, reduction of systemic oxidative stress and macrophage infiltration. Investigation of molecular mechanisms in bone marrow (BM)-derived progenitor cells from mice revealed that BMC therapy and, more consistently, in combination with MT ameliorated functional activity via decreased cellular senescence and increased telomerase and chemokine CXCR4 activities. Telomerase activity was also increased by HS alone or HS+MT and, more consistently, by BMC+HS alone or in combination with MT. CONCLUSIONS/INTERPRETATION: Intravenous autologous BMC and HS intervention together with MT increased therapeutic angiogenesis in the ApoE(-/-) diabetic mouse hindlimb.

Reprogramming of human somatic cells using human and animal oocytes.

There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc>4; p<0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.

The adaptor protein MyD88 is essential for E coli-induced preterm delivery in mice.

OBJECTIVE: We used a mouse model of infection-induced preterm delivery to examine the roles of 2 adaptor proteins with central functions in Toll-like receptor signaling: MyD88 (myeloid differentiation primary-response gene 88) and TRIF (Toll/IL-1 receptor (TIR)-domain-containing adaptor protein-inducing IFN-beta). STUDY DESIGN: Mice deficient (KO) for MyD88, TRIF, both (DKO) or neither (WT) were inoculated into the uterus with killed Escherichia coli. Delivery outcomes, fetal status, serum progesterone, and nuclear translocation of the transcription factor nuclear factor kappa B (NFkappaB) were determined. RESULTS: Preterm birth (delivery in less than 48 hours) occurred in WT and TRIF-KO animals in a dose-dependent fashion, reaching 100% with 5-10 x 10(9) bacteria, while MyD88-KO and DKO animals were completely protected from delivery. Intrauterine fetal survival, maintenance of circulating progesterone levels, and nuclear translocation of NFkappaB were also dependent upon MyD88 but not TRIF. In contrast, induction of uterine interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha) depends upon actions of both MyD88 and TRIF. CONCLUSION: E coli-induced preterm delivery in the mouse is completely dependent upon MyD88 but not TRIF.

Hemangioblasts from human embryonic stem cells generate multilayered blood vessels with functional smooth muscle cells.

BACKGROUND: The formation and regeneration of functional vasculatures require both endothelial cells (ECs) and vascular smooth muscle cells (SMCs). Identification and isolation of progenitors with potential for both EC and SMC lineage differentiation from an inexhaustible source, such as human embryonic stem (hES) or induced pluripotent stem cells, will be desirable for cell replacement therapy. METHOD: Recently, we have developed a serum-free and animal feeder-free differentiation system to generate blast cells (BCs) from hESCs. These cells possess the characteristics of hemangioblasts in vitro and are capable of repairing damaged retinal vasculatures, restoring blood flow in hind-limb ischemia and reducing the mortality rate after myocardial infarction in vivo. We demonstrate here that BCs express markers of SMCs and differentiate into smooth muscle-like cells (SMLCs), in addition to ECs and hematopoietic cells. RESULTS: When BCs from individual blast colonies were cultured in SMC medium, they differentiated into both ECs and SMLCs, which formed capillary-vascular-like structures after replating on Matrigeltrade mark. The SMLCs expressed SMC-specific markers (alpha-SM actin and calponin) and contracted upon treatment with carbachol. When implanted in nude mice, these cells formed microvasculature with ECs in Matrigel plaques. The BCs differentiated into both ECs and SMLCs, and incorporated into blood vessels after injection into ischemic tissue. CONCLUSION: These results demonstrate that hemangioblasts (BCs) generated from hESCs are tripotential and can provide a potentially inexhaustible source of cells for the treatment of human blood and vascular diseases.

Robust generation of hemangioblastic progenitors from human embryonic stem cells.

BACKGROUND: Human embryonic stem cells (hESCs) are a potentially inexhaustible source of cells for replacement therapy. However, successful preclinical and clinical progress requires efficient and controlled differentiation towards the specific differentiated cell fate. METHODS: We previously developed a strategy to generate blast cells (BCs) from hESCs that were capable of differentiating into vascular structures as well as into all hematopoietic cell lineages. Although the BCs were shown to repair damaged vasculature in multiple animal models, the large-scale generation of cells under these conditions was challenging. Here we report a simpler and more efficient method for robust generation of hemangioblastic progenitors. RESULTS: In addition to eliminating several expensive factors that are unnecessary, we demonstrate that bone morphogenetic protein (BMP)-4 and VEGF are necessary and sufficient to induce hemangioblastic commitment and development from hESCs during early stages of differentiation. BMP-4 and VEGF significantly upregulate T-brachyury, KDR, CD31 and Lmo2 gene expression, while dramatically downregulating Oct-4 expression. The addition of basic FGF during growth and expansion was found to further enhance BC development, consistently generating approximately 1 x 10(8) BCs from one six well plate of hESCs. CONCLUSION: This new method represents a significantly improved system for generating hemangioblasts from hESCs, and although simplified, results in an eightfold increase in cell yield.

GeneChip analysis of human embryonic stem cell differentiation into hemangioblasts: an in silico dissection of mixed phenotypes.

BACKGROUND: Microarrays are being used to understand human embryonic stem cell (hESC) differentiation. Most differentiation protocols use a multi-stage approach that induces commitment along a particular lineage. Therefore, each stage represents a more mature and less heterogeneous phenotype. Thus, characterizing the heterogeneous progenitor populations upon differentiation are of increasing importance. Here we describe a novel method of data analysis using a recently developed differentiation protocol involving the formation of functional hemangioblasts from hESCs. Blast cells are multipotent and can differentiate into multiple lineages of hematopoeitic cells (erythroid, granulocyte and macrophage), endothelial and smooth muscle cells. RESULTS: Large-scale transcriptional analysis was performed at distinct time points of hESC differentiation (undifferentiated hESCs, embryoid bodies, and blast cells, the last of which generates both hematopoietic and endothelial progenies). Identifying genes enriched in blast cells relative to hESCs revealed a genetic signature indicative of erythroblasts, suggesting that erythroblasts are the predominant cell type in the blast cell population. Because of the heterogeneity of blast cells, numerous comparisons were made to publicly available data sets in silico, some of which blast cells are capable of differentiating into, to assess and characterize the blast cell population. Biologically relevant comparisons masked particular genetic signatures within the heterogeneous population and identified genetic signatures indicating the presence of endothelia, cardiomyocytes, and hematopoietic lineages in the blast cell population. CONCLUSION: The significance of this microarray study is in its ability to assess and identify cellular populations within a heterogeneous population through biologically relevant in silico comparisons of publicly available data sets. In conclusion, multiple in silico comparisons were necessary to characterize tissue-specific genetic signatures within a heterogeneous hemangioblast population.

Recombinant HoxB4 fusion proteins enhance hematopoietic differentiation of human embryonic stem cells.

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.

Activation of toll-like receptors 2 or 3 and preterm delivery in the mouse.

The objective of this study is to test whether the activation of toll-like receptors (TLRs) 2 and 3 (innate immune receptors for gram-positive and viral pathogens, respectively) can induce preterm delivery. One uterine horn of preterm pregnant CD-1 mice at approximately 75% of gestation was injected with TLR-2 ligands (lipoteichoic acid [LTA] or peptidoglycan [PGN]) or the TLR-3 ligand polyinosinic:cytidylic acid (poly[I:C]). Preterm delivery was recorded. In a separate group of mice, tissue mRNAs were quantified by reverse transcriptase polymerase chain reaction 5 hours after treatment with PGN or poly(I:C). Intrauterine PGN and LTA induced preterm delivery, reaching 100% at maximal doses. Intraperitoneal PGN also induced preterm delivery but at lower rates (maximum = 55%). Intrauterine poly(I:C) induced preterm birth in up to 31% of mice. Poly(I:C) induced uterine interferon beta and chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES) but not interleukin 1beta, tumor necrosis factor, or lipopolysaccharide-induced CXC chemokine. PGN did not alter these mRNAs when compared with saline. Neither treatment induced gene expression in fetal membranes. Activation of either TLR-2 or -3 can induce preterm delivery in the mouse. Activation of TLR-3 with poly(I:C) induces interferon beta and the chemokine CCL5 in uterine tissues but not in fetal membranes.