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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., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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.

The pregnancy initiation and maintenance rates of nuclear transfer embryos produced from several bovine cell types were measured to determine which cell types produced healthy calves and had growth characteristics that would allow for genetic manipulation. Considerable variability between cell types from one animal and the same cell type from different animals was observed. In general, cultured fetal cells performed better with respect to pregnancy initiation and calving than adult cells with the exception of cumulous cells, which produced the highest overall pregnancy and calving rates. The cell type that combined relatively high pregnancy initiation and calving rates with growth characteristics that allowed for extended proliferation in culture were fetal genital ridge (GR) cells. Cultured GR cells used in nuclear transfer and embryo transfer initiated pregnancies in 40% of recipient heifers (197), and of all recipients that received nuclear transfer embryos, 9% produced live calves. Cultured GR cells doubled as many as 85 times overall and up to 75 times after dilution to single-cell culture. A comparison between transfected and nontransfected cells showed that transfected cells had lower pregnancy initiation (22% versus 32%) and calving (3.4% versus 8.9%) rates.

Central to the success of large animal cloning is the production of healthy animals that can provide products for human health, food, and other animal agriculture applications. We report development of cloned cattle derived from 34 genetically unique, nonembryonic cell lines using nuclear transfer performed between 1 January 1998 and 29 February 2000. Nearly 25% (535/2170) of the recipients receiving reconstructed embryos initiated pregnancy. Overall, 19.8% (106/535) of the initiated pregnancies resulted in live births, while 77% (82/106) of these cattle clones remain healthy and productive today. Although a wide variation in birth weight of clone calves was observed, their growth rates, reproductive performance, and lactation characteristics are similar to that found in noncloned dairy cattle. Our data represent the most comprehensive information on cattle derived from nuclear transfer procedures and indicate that this emerging reproductive technology offers unique opportunities to meet critical needs in both human health care and agriculture.

Nuclear transfer (NT) procedures were used to determine the in vivo developmental capacity of bovine embryonic cell lines derived from both morula- and blastocyst-stage embryos. These cell lines differed in morphology from trophoblast and endoderm-like cells. Regardless of initial donor embryo stage, cells in the resulting bovine embryonic cell lines had a small cytoplasmic/nuclear volume ratio and contained cytoplasmic vesicles. Developmental rates to blastocyst stage for NT embryos were improved when smaller cells (15 microns) rather than larger cells (18 microns or 21 microns) were used in the NT procedure and the recipient oocyte was activated after the cell fusion step. NT embryos produced from these embryonic cell lines, both morula- and blastocyst-derived, initiated pregnancies following transfer into recipient females. However, all of these pregnancies were lost prior to 60 days of gestation. These NT embryos were able to direct development through organogenesis, with one NT fetus reaching 55 days before death. When viable NT embryos were recovered during early gestation (38 days), an absence of cotyledons and a hemorrhagic response in the caruncles were observed. A chimera produced by aggregating an NT embryo with two 8-cell-stage blastomeres from in vitro-produced embryos developed through the 85th day of gestation. However, this conceptus was also deficient of cotyledons. DNA markers indicated that 50% of the chimera conceptus tissues were derived from the embryonic cell line. Blastocyst- and morula-derived embryonic cell line nuclei are pluripotent in that they can direct development through organogenesis, with subsequent pregnancy loss due, at least in part, to a deficiency in placentome development.