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 Protection by Sustained Nanoparticle Delivery of Oncostatin M and Ciliary Neurotrophic Factor Into Rodent Models of Retinal Degeneration.

Purpose: Retinitis pigmentosa (RP) is caused by mutations in more than 60 genes. Mutation-independent approaches to its treatment by exogeneous administration of neurotrophic factors that will preserve existing retinal anatomy and visual function are a rational strategy. Ciliary neurotrophic factor (CNTF) and oncostatin M (OSM) are two potent survival factors for neurons. However, growth factors degrade rapidly if administered directly. A sustained delivery of growth factors is required for translating their potential therapeutic benefit into patients. Methods: Stable and biocompatible nanoparticles (NP) that incorporated with CNTF and OSM (CNTF- and OSM-NP) were formulated. Both NP-trophic factors were tested in vitro using photoreceptor progenitor cells (PPC) and retinal ganglion progenitor cells (RGPC) derived from induced pluripotent stem cells and in vivo using an optic nerve crush model for glaucoma and the Royal College of Surgeons rat, model of RP (n = 8/treatment) by intravitreal delivery. Efficacy was evaluated by electroretinography and optokinetic response. Retinal histology and a whole mount analysis were performed at the end of experiments. Results: Significant prosurvival and pro-proliferation effects of both complexes were observed in both photoreceptor progenitor cells and RGPC in vitro. Importantly, significant RGC survival and preservation of vision and photoreceptors in both complex-treated animals were observed compared with control groups. Conclusions: These results demonstrate that NP-trophic factors are neuroprotective both in vitro and in vivo. A single intravitreal delivery of both NP-trophic factors offered neuroprotection in animal models of retinal degeneration. Translational Relevance: Sustained nanoparticle delivery of neurotrophic factors may offer beneficial effects in slowing down progressive retinal degenerative conditions, including retinitis pigmentosa, age-related macular degeneration, and glaucoma.

Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

AIM: To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). METHODS: We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. RESULTS: A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. CONCLUSION: Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

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.

Platelets generated from human embryonic stem cells are functional in vitro and in the microcirculation of living mice.

Platelets play an essential role in hemostasis and atherothrombosis. Owing to their short storage time, there is constant demand for this life-saving blood component. In this study, we report that it is feasible to generate functional megakaryocytes and platelets from human embryonic stem cells (hESCs) on a large scale. Differential-interference contrast and electron microscopy analyses showed that ultrastructural and morphological features of hESC-derived platelets were indistinguishable from those of normal blood platelets. In functional assays, hESC-derived platelets responded to thrombin stimulation, formed microaggregates, and facilitated clot formation/retraction in vitro. Live cell microscopy demonstrated that hESC-platelets formed lamellipodia and filopodia in response to thrombin activation, and tethered to each other as observed in normal blood. Using real-time intravital imaging with high-speed video microscopy, we have also shown that hESC-derived platelets contribute to developing thrombi at sites of laser-induced vascular injury in mice, providing the first evidence for in vivo functionality of hESC-derived platelets. These results represent an important step toward generating an unlimited supply of platelets for transfusion. Since platelets contain no genetic material, they are ideal candidates for early clinical translation involving human pluripotent stem cells.

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.