Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.

Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

Human embryonic stem cells.

The establishment of permanent human embryonic stem cell lines (hESCs) was first reported in 1998. Due to their pluripotent nature and ability to differentiate to all cell types in the body, they have been considered as a cell source for regenerative medicine. Since then, intensive studies have been carried out regarding factors regulating pluripotency and differentiation. hESCs are obtained from supernumerary human IVF (in vitro fertilization) embryos that cannot be used for the couple's infertility treatment. Today, we can establish and expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos. There are satisfactory tests for the demonstration of genetic stability, absence of tumorigenic mutations, functionality, and safety of hESCs. Clinical trials are ongoing for age-related macular degeneration (AMD) and spinal cord injury (SCI). This review focuses on the present state of these techniques.

Concise review: animal substance-free human embryonic stem cells aiming at clinical applications.

Human embryonic stem cells have been considered the gold standard as a cell source for regenerative medicine since they were first cultured in 1998. They are pluripotent and can form principally all the cells types in the body. They are obtained from supernumerary human in vitro fertilization embryos that cannot be used for infertility treatment. Following studies on factors regulating pluripotency and differentiation, we now have techniques to establish and effectively expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos in chemically defined feeder-free cultures. The genetic stability and absence of tumorigenic mutations can be determined. There are satisfactory animal tests for functionality and safety. The first clinical trials are ongoing for two indications: age-related macular degeneration and spinal cord injury.

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Costimulation blockade induces foxp3(+) regulatory T cells to human embryonic stem cells.

Transplantation of human embryonic stem cells (hESCs), like other allogeneic cellular transplants, require immunomodulation or immunosuppression in order to be maintained in the recipient. Costimulation blockade applied at the time of transplantation inhibits costimulatory signals in the immunological synapse leading to a state of anergy in the donor reactive T-cell population and a state of immunological tolerance in the host. In models of solid organ transplantation, tolerance is maintained by the infiltration of Foxp3(+) regulatory T cells into the graft. In order to study if regulatory T cells could be generated to hESC transplants, costimulation blockade (CTLA4Ig, anti-CD40L, anti-LFA-1) was administered for the first week after transplantation of two different hESC lines implanted under the kidney capsule of wild-type mice. hESC transplants were maintained indefinitely, and when harvested at long-term follow-up, Foxp3(+) T-cells were found surrounding the graft, implying the maintenance of tolerance through the induction of regulatory T cells. These results imply that costimulation blockade could be a useful treatment strategy for the induction of tolerance to hESC transplants and may down-modulate immune responses locally around the graft.

Dual R3R5 tropism characterizes cerebrospinal fluid HIV-1 isolates from individuals with high cerebrospinal fluid viral load.

OBJECTIVE: To study the use of major and alternative coreceptors by HIV-1 isolates obtained from paired plasma and cerebrospinal fluid (CSF) samples. DESIGN: Paired plasma and CSF isolates from HIV-1-infected individuals with varying clinical, virologic, and immunologic parameters were assessed for the ability to infect indicator cells expressing a panel of coreceptors with documented expression in the central nervous system (CNS). METHODS: HIV-1 isolates obtained from plasma and CSF in 28 individuals with varying viral load, CD4 T-cell counts, and with or without AIDS-defining disease were analyzed for the ability to infect NP2.CD4 cells stably expressing a panel of HIV coreceptors (CCR5, CXCR4, CCR3, CXCR6, GPR1, APJ, ChemR23, RDC-1 or BLT1). RESULTS: All isolates from both plasma and CSF utilized CCR5 and/or CXCR4. However, the ability to use both CCR3 and CCR5 (R3R5) was more pronounced in CSF isolates and correlated with high CSF viral load and low CD4 T-cell count. Notably, four out of five CSF isolates of subtype C origin exhibited CXCR6 use, which coincided with high CSF viral load despite preserved CD4 T-cell counts. The use of other alternative coreceptors was less pronounced. CONCLUSION: Dual-tropic R3R5 HIV-1 isolates in CSF coincide with high CSF viral load and low CD4 T-cell counts. Frequent CXCR6 use by CSF-derived subtype C isolates indicates that subtype-specific differences in coreceptor use may exist that will not be acknowledged when assessing plasma virus isolates. The findings may also bare relevance for HIV-1 replication within the CNS, and consequently, for the neuropathogenesis of AIDS.

Mode of coreceptor use by R5 HIV type 1 correlates with disease stage: a study of paired plasma and cerebrospinal fluid isolates.

Through the use of chimeric CXCR4/CCR5 receptors we have previously shown that CCR5-tropic (R5) HIV-1 isolates acquire a more flexible receptor use over time, and that this links to a reduced viral susceptibility to inhibition by the CCR5 ligand RANTES. These findings may have relevance with regards to the efficacy of antiretroviral compounds that target CCR5/virus interactions. Compartmentalized discrepancies in coreceptor use may occur, which could also affect the efficacy of these compounds at specific anatomical sites, such as within the CNS. In this cross-sectional study we have used wild-type CCR5 and CXCR4 as well as chimeric CXCR4/CCR5 receptors to characterize coreceptor use by paired plasma and cerebrospinal fluid (CSF) isolates from 28 HIV-1-infected individuals. Furthermore, selected R5 isolates, with varying chimeric receptor use, were tested for sensitivity to inhibition by the CCR5 antagonist TAK-779. Discordant CSF/plasma virus coreceptor use was found in 10/28 patients. Low CD4+ T cell counts correlated strongly with a more flexible mode of R5 virus CCR5 usage, as disclosed by an increased ability to utilize chimeric CXCR4/CCR5 receptors, specifically receptor FC-2. Importantly, an elevated ability to utilize chimeric receptors correlated with a reduced susceptibility to inhibition by TAK-779. Our findings show that a discordant CSF and plasma virus coreceptor use is not uncommon. Furthermore, we provide support for an emerging paradigm, where the acquisition of a more flexible mode of CCR5 usage is a key event in R5 virus pathogenesis. This may, in turn, negatively impact the efficacy of CCR5 antagonist treatment in late stage HIV-1 disease.

HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children.

BACKGROUND: HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad) viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression. METHODOLOGY/PRINCIPAL FINDINGS: Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow) phenotype (n = 20), but R5(broad) and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad) and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad) phenotype, however, the presence of the R5(broad) virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad) viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad) phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. CONCLUSIONS/SIGNIFICANCE: Our results show that R5(broad) viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow) phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.

Biological and genetic evolution of HIV type 1 in two siblings with different patterns of disease progression.

To investigate the immunological and virological factors that may lead to different patterns of disease progression characteristic of HIV-1-infected children, two HIV-1-infected siblings, a slow and a fast progressor, were followed prospectively before the onset of highly active antiretroviral therapy. Viral coreceptor usage, including the use of CCR5/CXCR4 chimeric receptors, macrophage tropism, and sensitivity to the CC-chemokine RANTES, has been studied. An autologous and heterologous neutralizing antibody response has been documented using peripheral blood mononuclear cells- and GHOST(3) cell line-based assays. Viral evolution was investigated by env C2-V3 region sequence analysis. Although both siblings were infected with HIV-1 of the R5 phenotype, their viruses showed important biological differences. In the fast progressor there was a higher RANTES sensitivity of the early virus, an increased trend to change the mode of CCR5 receptor use, and a larger genetic evolution. Both children developed an autologous neutralizing antibody response starting from the second year with evidence of the continuous emergence of resistant variants. A marked viral genetic and phenotypic evolution was documented in the fast progressor sibling, which is accompanied by a high viral RANTES sensitivity and persistent neutralizing antibodies.

CCR5 N-terminal region plays a critical role in HIV-1 inhibition by Toxoplasma gondii-derived cyclophilin-18.

Molecular mimicry of chemokine ligands has been described for several pathogens. Toxoplasma gondii produces a protein, cyclophilin-18 (C-18), which binds to the human immunodeficiency virus (HIV) co-receptor CCR5 and inhibits fusion and infection of T cells and macrophages by R5 viruses but not by X4 viruses. We recently identified structural determinants of C-18 required for anti-HIV activity (Yarovinsky, F., Andersen, J. F., King, L. R., Caspar, P., Aliberti, J., Golding, H., and Sher, A. (2004) J. Biol. Chem. 279, 53635-53642). Here we have elucidated the fine specificity of CCR5 residues involved in binding and HIV inhibitory potential of C-18. To delineate the regions of CCR5 involved in C-18 binding, we analyzed C-18 inhibition of cells expressing CXCR4/CCR5 chimeric receptors and CCR5 with a truncated N terminus (Delta2-19). These experiments identified a critical role for the N terminus of CCR5 in C-18 binding and anti-HIV activity. Studies with a large panel of CCR5 N-terminal peptides, including Tyr-sulfated analogues, truncated peptides, and alanine-scanning mutants, suggested that each of the 12-17 amino acids in the N terminus of CCR5 are essential for C-18 binding and inhibitory activity. Tyr sulfation did not improve C-18 reactivity. This finding is of interest because the same CCR5 N-terminal region was shown previously to play a key role in binding of HIV-1 envelope glycoproteins. The elucidation of the functional C-18-binding mechanism may help in the rational design of novel antiviral agents against HIV.

Coevolution of RANTES sensitivity and mode of CCR5 receptor use by human immunodeficiency virus type 1 of the R5 phenotype.

The evolution of human immunodeficiency virus type 1 (HIV-1) coreceptor use has been described as the acquisition of CXCR4 use linked to accelerated disease progression. However, CXCR4-using virus can be isolated only from approximately one-half of individuals with progressive HIV-1 disease. The other half continue to yield only CCR5-using viruses (R5 phenotype) throughout the course of disease. In the present work, the use of receptor chimeras between CCR5 and CXCR4 allowed us to study the evolution of HIV-1 with the R5 phenotype, which was not revealed by studies of wild-type coreceptor use. All together, 246 isolates (173 with the R5 phenotype) from 31 individuals were tested for their ability to infect cells through receptor chimeras. R5(narrow) virus was able to use only wild-type CCR5, whereas R5(broad(1)) to R5(broad(3)) viruses were able to use one to three chimeric receptors, respectively. Broad use of chimeric receptors was interpreted as an increased flexibility in the mode of receptor use. R5(broad) isolates showed higher infectivity in cells expressing wild-type CCR5 than R5(narrow) isolates. Also, the increased flexibility of R5(broad) isolates was concomitant with a lower sensitivity to inhibition by the CC chemokine RANTES. Our results indicate a close relationship between HIV-1 phenotypic changes and the pathogenic process, since the mode and efficiency of CCR5 use as well as the decrease in the RANTES sensitivities of isolated viruses are significantly correlated with CD4(+)-T-cell decline in a patient. One possible explanation is that ligand competition at the CCR5 receptor or changed CCR5 availability may shape the outcome of HIV-1 infection.

HIV biological variability unveiled: frequent isolations and chimeric receptors reveal unprecedented variation of coreceptor use.

OBJECTIVE: To follow the evolution of coreceptor use in HIV-1-infected individuals with varying rates of disease progression. METHODS: The coreceptor use of 278 sequential HIV-1 isolates from 23 individuals was tested by infection of two human cell lines, U87.CD4 and GHOST(3), expressing CD4 and CCR1, CCR2b, CCR3, CCR5, CXCR4, CXCR6 or BOB. Differences in coreceptor use were further dissected by testing chimeric coreceptors constructed by exchanging successively larger parts of CCR5 for corresponding regions of CXCR4. RESULTS: Three patterns of coreceptor use were distinguished: no evolution, evolution to CXCR4 use, and fluctuation. No evolution with stable CCR5 use (R5 phenotype) was linked to slow progression over 8-10 years in four patients. CXCR4-using virus was present from the onset (five patients) or appeared during clinical progression in all other patients, while taking zidovudine or didanosine monotherapy. The fluctuating pattern of coreceptor use, defined as reappearance of virus with R5 phenotype, was observed in five patients and, interestingly, followed initiation of highly active antiretroviral therapy in three of these. Monotropic R5 or X4 viruses were more selective in chimeric receptor use than R5X4 or multitropic viruses. Most importantly, the efficiency of chimeric receptor use increased over time. CONCLUSION: The increase in efficiency of chimeric receptor use allows a new interpretation of evolution of HIV-1 coreceptor use. Evolution could be a continuous process that may lead to changes in the way a coreceptor is used, with the potential of profound alteration in signalling at that receptor.

Molecular mapping of epitopes for interaction of HIV-1 as well as natural ligands with the chemokine receptors, CCR5 and CXCR4.

OBJECTIVE: Mapping coreceptor epitopes used by the prototypic R5 and X4 strains, HIV-1BaL and HIV-1IIIB, in comparison with epitopes involved in the activation and signaling induced by the natural ligands, RANTES and SDF-1beta. DESIGN: Receptor hybrids between CCR5 and CXCR4 were constructed. METHODS: Using single-overlap and extension PCR, increasing portions of CCR5 were replaced with corresponding parts of CXCR4. Viral interaction with these constructs was monitored in infection experiments using stably transfected cell lines, and ligand-induced activation of cells transiently expressing the constructs was measured in terms of calcium fluxes. RESULTS: SDF-1beta required an essentially complete CXCR4, whereas RANTES demanded both the N terminus and the first two extracellular loops of CCR5. HIV-1 infection experiments emphasized the importance of the CCR5 N terminus for infection with HIV-1BaL, whereas HIV-1IIIB was less demanding in its use of CXCR4. CONCLUSION: This study, for the first time monitoring CCR5 and CXCR4 ligand activation and HIV-1 interaction concomitantly, indicates that ligands and virus use different receptor epitopes which, in turn, vary between the two receptors. One particular chimera (FC-4b), having its junctional region close to the conserved cysteine in ECL2, functioned as coreceptor for both HIV-1BaL and HIV-1IIIB, but was not activated with RANTES or SDF-1beta. The results provide a basis for tailoring drugs that block viral entry through the two major coreceptors without interfering with their physiological function.

Optimized reporter gene assays based on a synthetic multifunctional promoter and a secreted luciferase.

Efficient screening for ligands of seven-transmembrane, G-protein-coupled receptors, whether transfected or endogenously expressed, often involves cell-based reporter assays. Here we describe the development of reporter gene assays in HeLa cells. The reporter construct includes a synthetic multifunctional promoter with several different response motifs (NF-kappaB, STAT, and AP-1) and hence efficiently funnels several signaling pathways. The assay, performed with the resulting reporter cell line HFF11, has an exceptional high Z-factor and a large signal-to-background ratio. To facilitate cell handling during screening, we introduced a secreted Renilla luciferase as a reporter enzyme. HR36 reporter cells, equipped with the construct, were added to ligands present in a multiwell plate and after addition of coelenterazine they produced a luminescence readout. This procedure economizes cell handling and at the same time increases assay quality and sensitivity