Assessment of safety and immunogenicity of MHC homozygous iPSC-derived CD34+ hematopoietic progenitors in an NHP model.

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits.

Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos. Methods: DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis. Results: Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites. Discussion: This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.

Genome editing of CCR5 by CRISPR-Cas9 in Mauritian cynomolgus macaque embryos.

The discovery that CCR5 serves as an R5-HIV-1 co-receptor, coupled with findings of protection from HIV infection in individuals lacking CCR5, led to the exploration of novel therapeutic strategies for HIV infection based on genome editing of CCR5. Advancing translation of CCR5-mutant-based cellular therapies for HIV requires development of novel physiologically relevant animal models. Mauritian cynomolgus macaques (MCMs), with high degree of MHC allele sharing, are valuable models for HIV-1 research and stem cell therapies. To facilitate the generation of a CCR5-mutant MHC-defined MCM model, we explored editing the CCR5 gene in MCM embryos via CRISPR-Cas9. We refined ovarian stimulation and in vitro fertilization (IVF) methods established for Chinese cynomolgus macaques to generate in vitro MCM embryos. Time-lapse embryo imaging was performed to assess the timing of MCM embryonic developmental events in control and CRISPR-Cas9 microinjected embryos. Using a dual-guide gene targeting approach, biallelic deletions in the CCR5 gene were introduced into ~ 23-37% of MCM embryos. In addition, single blastomere PCR analysis revealed mosaicism in CCR5 editing within the same embryo. Successful development of IVF and CCR5 editing protocols in MCM embryos lays a foundation for the creation of CCR5-mutant MCMs to assess novel stem cell-based HIV therapeutics.

Cryopreservation of Mauritian Cynomolgus Macaque (Macaca fascicularis) Sperm in Chemically Defined Medium.

The objective of this study was to optimize cryopreservation of sperm from Mauritian cynomolgus macaques (MCM) in defined conditions. Sperm viability and motility were compared between sperm cryopreserved in chemically-defined freezing media with variable osmolarity and the presence of either ethylene glycol or glycerol. The highest percentage viability (after freeze-thaw) was seen in sperm samples that were cryopreserved in medium with an osmolarity of 310 mOsm, while higher osmolarities markedly decreased sperm viability. Ethylene glycol and glycerol at concentrations of 4.6% and 5%, respectively, preserved sperm viability to an equivalent degree. Although higher motility rates and higher straight-line velocities were observed in sperm samples frozen in glycerol compared with ethylene glycol, these differences were not statistically significant. Thawed sperm frozen in defined conditions with glycerol were capable of fertilizing MCM oocytes in vitro, with development to the blastocyst stage. The protocol described here provides an effective method for cryopreservation of sperm to facilitate subsequent in vitro fertilization and genome editing of embryos in MCM species.

Embryonic stem cells from morula.

It has been shown that it is possible to establish human embryonic stem cell (hESC) lines from morula. Details of the aforementioned injection method of morula under blastocyst are described in this chapter. This chapter also discloses the application of simultaneous staining for two markers, TRA-2-39 and Oct-4, for characteristics of nondifferentiated hESC derived from morula and gives a method. Technical approaches of freezing morula-derived hESC are discussed.

Reprogramming of human somatic cells by embryonic stem cell cytoplast.

Somatic cell nuclear transfer (SCNT) provides the basis for the development of patient-specific stem cell lines. Recent progress in SCNT suggested the presence of reprogramming factors in human embryonic stem (hES) cells, although no method is currently available for replacement of nuclei of hES cells by somatic cell nuclei. An original technique has been developed, involving the fusion of different types of somatic cells with hES cells, which allowed a complete replacement of the nuclei of hES cells by nuclei of somatic cells. The resulting 'cybrids' were demonstrated to have the genotype of the donor somatic cells and 'stemness' of the recipient hES cells. However, the colonies isolated from the resulting fusion contained a mixture of these cybrid cells with the cells with the recipient nuclei, as well as hybrid cells containing both donor and recipient nuclei, so future purification will be necessary before the technique can be considered for future practical application.

Cytoplasmic cell fusion: Stembrid technology for reprogramming pluripotentiality.

Recent progress in somatic cell nuclear transfer (SCNT) provides the evidence for the presence of reprogramming factors in human embryonic stem cells (hESC). Hybrid hESC with donor human somatic nuclei have been established, but the resulting hybrid hESC contained DNA originating from both hESC and donor somatic cells. There is still no method to completely replace the hESC nuclei by the nuclei of somatic cells and to obtain the pure colonies of hESC with donor genotype. We present here the original technology, which is based on enucleation of h ESC and their fusion with the adult somatic cells, resulting in the establishment of individual-specific hESC with the genotype of the donor somatic cells. The resulting constructs was demonstrated to have the "stemness" of hESC and the genotype of the donor somatic cells. So this "Stembrid technology," may be used for the construction of patient-specific hESC.

Morula-derived human embryonic stem cells.

Human embryonic stem (ES) cells are known to derive from the inner cell mass of blastocyst. Although the embryos of other developmental stages have also been used as a source for ES cells in animal models, the feasibility of obtaining ES cell lines from human morula is not known, despite being an obvious source available through assisted reproduction and preimplantation genetic diagnosis programmes. This study describes an original technique for derivation of ES cells from human morula, which enabled the establishment of eight morula-derived ES cell lines. These ES cell lines were shown to have no morphological differences from the ES cells derived from blastocysts, and expressed the same ES cell specific markers, including Oct-4, tumour-resistance antigens TRA-2-39, stage-specific embryonic antigens SSEA-3 and SSEA-4, and high molecular weight glycoproteins TRA-1-60 and TRA-1-81, detected in the same colony of morula-derived ES cells showing specific alkaline phosphatase expression. No differences were observed in these marker expressions in the morula-derived ES cells cultured in the feeder layer free medium. Similar to ES cell originating from blastocyst, the morula-derived ES cells were shown to spontaneously differentiate in vitro into a variety of cell types, including the neuron-like and contracting primitive cardiocyte-like cells.