Metabolic rescue in pluripotent cells from patients with mtDNA disease.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Mitochondrial replacement therapy in reproductive medicine.

Mitochondrial dysfunction is implicated in disease and age-related infertility. Mitochondrial replacement therapies (MRT) in oocytes or zygotes, such as pronuclear (PNT), spindle (ST), or polar body (PBT) transfer, could prevent second-generation transmission of mitochondrial DNA (mtDNA) defects. PNT, associated with high levels of mtDNA carryover in mice but low levels in human embryos, carries ethical issues secondary to donor embryo destruction. ST, developed in primates, supports normal development to adults and low mtDNA carryover. PBT in mice, coupled with PN or ST, may increase the yield of reconstructed embryos with low mtDNA carryover. MRT also offers replacement of the deficient cytoplasm in oocytes from older patients, with the expectation of high pregnancy rates following in vitro fertilization.

Coculture of monkey ovarian tissue increases survival after vitrification and slow-rate freezing.

OBJECTIVE: To assess whether coculture of monkey ovarian tissue after low-temperature storage enhances follicular viability. To assess a novel method of vitrifying ovarian tissue. DESIGN: Prospective in vitro study. SETTING: University-affiliated national research center. ANIMAL(S): Ovaries from 15 cynomolgus or rhesus macaques (1-11 years). INTERVENTION(S): Vitrification using a containerless liquid nitrogen emersion system that involves dropping thin cortical pieces suspended in cyroprotectant directly into liquid nitrogen with outcome compared with slow-rate-controlled freezing. Before analysis, some of the thawed tissue was cocultured on mitotically inactivated mouse fetal fibroblast monolayers supplemented with FSH, insulin, transferrin, and selenium. MAIN OUTCOME MEASURE(S): Percentage of oocytes viable using live-dead fluorescent staining with carboxyfluorescein diacetate succinimidyl ester and propidium iodide. RESULT(S): Postthaw survival rates were 70.4% +/- 4.8% of 1,705 follicles after vitrification and 67.3% +/- 1.9% of 1,895 follicles after slow-rate freeze in six trials with each method. Coculture of the thawed tissue increased the viability, respectively, to 89% +/- 2.1% of 2,833 follicles previously vitrified and to 90.3% +/- 1.9% of 2,109 follicles after a slow-rate freeze (P<.01). Primordial follicles (30- to 50-microm diameter) were the vast majority of surviving follicles after thaw and coculture. Follicular viability in control fresh tissue (eight trials) was 76.0% +/- 4.1%, suggesting negligible loss in follicular viability after cryopreservation. CONCLUSION(S): Coculture of thawed ovarian tissue on mouse fetal fibroblasts and FSH increases the percentage of viable follicles. A novel method of vitrifying ovarian tissue is as effective as slow-rate freezing. These approaches may improve graft survival and function when used to treat chemotherapy-induced sterility.

Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons.

We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERbeta), and progestin receptor (PR), but not estrogen receptor-alpha (ERalpha). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A-autoreceptor, ERbeta, and PR in the differentiated population. A low level of expression of ERalpha mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.

Collection and fertilization potential of sperm from the Sulawesi crested black macaque (Macaca nigra).

In this study, semen was obtained by rectal probe electrostimulation (RPE) from the Sulawesi crested black macaque (Macaca nigra). Three experimental series were conducted. First, semen was collected from four animals anesthetized with either tiletamine-zolazepam (Telazol®) or ket-amine-HCl (Vetalar®) (five collections from each animal with each drug). Because of greater muscle relaxation and analgesia, we found tiletaminezolazepam to be an attractive alternative to ketamine-HCl as an anesthetic agent for RPE in M. nigra. Second, semen was collected from another four animals at stimulation frequencies of either 30 Hz or 60 Hz (five collections from each animal at each frequency). There were no significant differences in sperm number, in percentage of sperm with progressive motility, in the current required for sample recovery between tiletamine-zolazepam or ketamine-HCl anesthesia, or between a 30 Hz or 60 Hz stimulation frequency. Third, to check for retrograde sperm loss, the bladders of four animals were emptied, flushed with sterile saline, and then infused with TALP-Hepes medium. After RPE, sperm numbers in the bladder were compared with those in the ejaculate. Although sperm were recovered from the bladder [1.6 (± 0.9) × 106] (mean ± SEM), the numbers were significantly less (P < 0.05) than those in the ejaculate [49 (± 18) × 106]. The percentage of sperm with normal morphology in these samples was high (96.8 ± 1.0%). The average sperm number in the 84 samples collected for this study was 33.8 (± 4.1) × 106. In preliminary experiments, we found that M. nigra sperm will fertilize rhesus monkey oocytes (Macaca mulatta) in vitro. © 1992 Wiley-Liss, Inc.