Survival of Neural Progenitors Derived from Human Embryonic Stem Cells Following Subretinal Transplantation in Rodents.

Purpose: To examine the survival of neural progenitors (NPs) cells derived from human embryonic stem cells (hESCs) following subretinal (SR) transplantation in rodents. Methods: hESCs engineered to express enhanced green fluorescent protein (eGFP) were differentiated in vitro toward an NP fate using a 4-week protocol. State of differentiation was characterized by quantitative-PCR. NPs in suspension (75,000/µl) were transplanted to the SR-space of Royal College of Surgeons (RCS) rats (n = 66), nude-RCS rats (n = 18), and NOD scid gamma (NSG) mice (n = 53). Success of engraftment was determined at 4 weeks post-transplant by in vivo visualization of GFP-expression using a properly filtered rodent fundus camera. Transplanted eyes were examined in vivo at set time points using the fundus camera, and in select cases, by optical coherence tomography imaging, and after enucleation, by retinal histology and immunohistochemistry. Results: In RCS rats, cell rejection was observed in 29% of eyes at 6 weeks, rising to 92% at 8 weeks. In the more immunodeficient nude-RCS rats, the rejection rate was still high reaching 62% of eyes at 6 weeks post-transplant. Following transplantation in highly immunodeficient NSG mice, survival of the hESC-derived NPs was much improved, with 100% survival at 9 weeks and 72% at 20 weeks. A small number of eyes that were followed past 20 weeks showed survival also at 22 weeks. Conclusions: Immune status of recipient animals influences transplant survival. Highly immunodeficient NSG mice provide a better model for studying long-term survival, differentiation, and possible integration of hESC-derived NPs. Clinical Trial Registration numbers: NCT02286089, NCT05626114.

Hadassah, provider of "Regulatory-Ready" pluripotent clinical-grade stem cell banks.

The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines.

Standardization of the teratoma assay for analysis of pluripotency of human ES cells and biosafety of their differentiated progeny.

Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.

Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension.

Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.

Directed differentiation of human embryonic stem cells into functional retinal pigment epithelium cells.

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

Rational design of a selective antagonist of epsilon protein kinase C derived from the selective allosteric agonist, pseudo-RACK peptide.

We have previously shown that domains involved in binding of protein kinase C (PKC) isozymes to their respective anchoring proteins (RACKs) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named psiRACK peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the psiRACK sequences within each PKC isozyme have at least one non-homologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the psiRACK sequence. We focused on epsilonPKC psiRACK peptide, where the pseudo-epsilonRACK sequence (psiepsilonRACK; HDAPIGYD; corresponding to epsilonPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to epsilonRACK285-292) in the second amino acid. Here we show that changing the charge of the psiepsilonRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.

Whole genome analysis of human neural stem cells derived from embryonic stem cells and stem and progenitor cells isolated from fetal tissue.

Multipotent neural stem cells (NSC) have been derived from human embryonic stem cells (hESC) as well as isolated from fetal tissues. However, there have been few exclusive markers of NSC identified to date, and the differences between NSC from various sources are poorly understood. Although cells isolated from these two sources share many important characteristics, it is not clear how closely they are related in terms of gene expression. Here, we compare the gene expression profiles of 11 lines of NSC derived from hESC (ES_NSC), four lines of NSC isolated from fetus (F_NSC), and two lines of restricted progenitors in order to characterize these cell populations and identify differences between NSC derived from these two sources. We showed that ES_NSC were clustered together with high transcriptional similarities but were distinguished from F_NSC, oligodendrocyte precursor cells, and astrocyte precursor cells. There were 17 genes expressed in both ES_NSC and F_NSC whose expression was not identified in restricted neural progenitors. Furthermore, the major differences between ES_NSC and F_NSC were mostly observed in genes related to the key neural differentiation pathways. Here, we show that comparison of global gene expression profiles of ES_NSC, F_NSC, and restricted neural progenitor cells makes it possible to identify some of the common characteristics of NSC and differences between similar stem cell populations derived from hESCs or isolated from fetal tissue. Disclosure of potential conflicts of interest is found at the end of this article.

Transplantation of human embryonic stem cell-derived neural progenitors improves behavioral deficit in Parkinsonian rats.

Human embryonic stem cells (hESCs) may potentially serve as a renewable source of cells for transplantation. In Parkinson's disease, hESC-derived dopaminergic (DA) neurons may replace the degenerated neurons in the brain. Here, we generated highly enriched cultures of neural progenitors from hESCs and grafted the progenitors into the striatum of Parkinsonian rats. The grafts survived for at least 12 weeks, the transplanted cells stopped proliferating, and teratomas were not observed. The grafted cells differentiated in vivo into DA neurons, though at a low prevalence similar to that observed following spontaneous differentiation in vitro. Transplanted rats exhibited a significant partial correction of D-amphetamine and apomorphine-induced rotational behavior, along with a significant improvement in stepping and placing non-pharmacological behavioral tests. While transplantation of uncommitted hESC-derived neural progenitors induced partial behavioral recovery, our data indicate that the host-lesioned striatum could not direct the transplanted neural progenitors to acquire a dopaminergic fate. Hence, induction of their differentiation toward a midbrain fate prior to transplantation is probably required for complete correction of behavioral deficit. Our observations encourage further developments for the potential use of hESCs in the treatment of Parkinson's disease.

State-specific monoclonal antibodies identify an intermediate state in epsilon protein kinase C activation.

Evaluation of the activation state of protein kinase C (PKC) isozymes relies on analysis of subcellular translocation. A monoclonal antibody, 14E6, specific for the activated conformation of epsilonPKC, was raised using the first variable (V1) domain of epsilonPKC as the immunogen. 14E6 binding is specific for epsilonPKC and is greatly increased in the presence of PKC activators. Immunofluorescence staining by 14E6 of neonatal rat primary cardiac myocytes and the NG108-15 neuroblastoma glioma cell line, NG108-15/D2, increases rapidly following cell activation and is localized to new subcellular sites. However, staining of translocated epsilonPKC with 14E6 is transient, and the epitope disappears 30 min after activation of NG-108/15 cells by a D2 receptor agonist. In contrast, subcellular localization associated with activation, as determined by commercially available polyclonal antibodies, persists for at least 30 min. In vitro, epsilonRACK, the receptor for activated epsilonPKC, inhibits 14E6 binding to epsilonPKC, suggesting that the 14E6 epitope is lost or hidden when active epsilonPKC binds to its RACK. Therefore, the 14E6 antibody appears to identify a transient state of activated but non-anchored epsilonPKC. Moreover, binding of 14E6 to epsilonPKC only after activation suggests that lipid-dependent conformational changes associated with epsilonPKC activation precede binding of the activated isozyme to its specific RACK, epsilonRACK. Further, monoclonal antibody 14E6 should be a powerful tool to study the pathways that control rapid translocation of epsilonPKC from cytosolic to membrane localization on activation.