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Don P Wolf

Publications and source records attributed to Don P Wolf.

At least 19 recordsLinked to original sources
PubMed2006

Transcriptional profiling of rhesus monkey embryonic stem cells.

Embryonic stem cells (ESCs) may be able to cure or alleviate the symptoms of various degenerative diseases. However, unresolved issues regarding survival, functionality, and tumor formation mean a prudent approach should be adopted towards advancing ESCs into human clinical trials. The rhesus monkey provides an ideal model organism for developing strategies to prevent immune rejection and test the feasibility, safety, and efficacy of ESC-based medical treatments. Transcriptional profiling of rhesus monkey ESCs provides a foundation for pre-clinical ESC research in this species. In the present study, we used microarray technology, immunocytochemistry, reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR) to characterize and transcriptionally profile rhesus monkey ESCs. We identified 367 stemness gene candidates that were highly (>85%) conserved across five different ESC lines. Rhesus monkey ESC lines maintained a pluripotent undifferentiated state over a wide range of POU5F1 (also known as OCT4) expression levels, and comparisons between rhesus monkey, mouse, and human stemness genes revealed five mammalian stemness genes: CCNB1, GDF3, LEFTB, POU5F1, and NANOG. These five mammalian genes are strongly expressed in rhesus monkey, mouse, and human ESCs, albeit only in the undifferentiated state, and represent the core key mammalian stemness factors.

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PubMed2006

Nuclear transfer in nonhuman primates.

The nonhuman primate is a highly relevant model for the study of human diseases, and currently there is a significant need for populations of animals with specific genotypes that can not be satisfied by the capture of animals from the wild or by conventional breeding. There is an even greater need for genetically identical animals in vaccine development or tissue transplantation research, where immune system function is under study. Efficient somatic cell nuclear transfer (SCNT) procedures could provide a source for genetically identical nonhuman primates for biomedical research. SCNT offers the possibility of cloning animals using cultured cells and potentially provides an alternative approach for the genetic modification of primates. The opportunity to introduce precise genetic modifications into cultured cells by gene targeting procedures, and then use these cells as nuclear donors in SCNT, has potential application in the production of loss-of-function monkey models of human diseases. We were initially successful in producing monkeys by NT using embryonic blastomeres as the source of donor nuclei and have repeated that success. However, when somatic cells are used as nuclear donor cells, the developmental potential of monkey SCNT embryos is limited, and somatic cell cloning has not yet been accomplished in primates. High rates of in vitro development to blastocysts, comparable with in vitro fertilization controls, and successful production of rhesus monkeys by NT from embryonic blastomeres suggests that basic cloning procedures, including enucleation, fusion, and activation, are consistent with the production of viable embryos. Although modifications or additional steps in SCNT are clearly warranted, the basic procedures will likely be similar to those extant for embryonic cell NT. In this chapter, we describe detailed protocols for rhesus macaque embryonic cell NT, including oocyte and embryo production, micromanipulation, and embryo transfer in nonhuman primates.

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PubMed2005

Aberrant genomic imprinting in rhesus monkey embryonic stem cells.

Genomic imprinting involves modification of a gene or a chromosomal region that results in the differential expression of parental alleles. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. Additionally, abnormal imprinting occurs in mouse embryonic stem cells (ESCs) and in clonally derived animals. Imprinted gene expression patterns in primate ESCs are largely unknown, despite the clinical potential of the latter in the cell-based treatment of human disease. Because of the possible implications of abnormal gene expression to cell or tissue replacement therapies involving ESCs, we examined allele specific expression of four imprinted genes in the rhesus macaque. Genomic and complementary DNA from embryos and ESC lines containing useful single nucleotide polymorphisms were subjected to polymerase chain reaction-based amplification and sequence analysis. In blastocysts, NDN expression was variable indicating abnormal or incomplete imprinting whereas IGF2 and SNRPN were expressed exclusively from the paternal allele and H19 from the maternal allele as expected. In ESCs, both NDN and SNRPN were expressed from the paternal allele while IGF2 and H19 showed loss of imprinting and biallelic expression. In differentiated ESC progeny, these expression patterns were maintained. The implications of aberrant imprinted gene expression to ESC differentiation in vitro and on ESC-derived cell function in vivo after transplantation are unknown.

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PubMed2005

Transplantable neural progenitor populations derived from rhesus monkey embryonic stem cells.

Cell-based therapies using embryonic stem cells (ESCs) in the treatment of neural disease will require the generation of homogenous donor neural progenitor (NP) populations. Here we describe an efficient culture system containing hepatocyte growth factor (HGF) and G5 supplement for the production of highly enriched (88.3%+/-8.1%) populations of NPs from rhesus monkey ESCs. Additional purification resulted in NP preparations that were 98% nestin positive. Moreover, NPs, as monolayers or neurospheres, could be maintained for prolonged periods of time in media containing HGF+G5 or G5 alone. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes, and oligodendrocytes. The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, approximately 25% of transplanted NPs survived and 65%-80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. Subcloning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro and in vivo in rat brains. These observations set the stage for obtaining highly enriched NPs and evaluating the efficacy of NP-based transplantation therapy in the nonhuman primate and will provide a platform for probing the molecular mechanisms that control neural induction.

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PubMed2005

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.

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PubMed2005

Low temperature storage of rhesus monkey spermatozoa and fertility evaluation by intracytoplasmic injection.

The objective was to develop a sperm freezing procedure suitable for use in the propagation of valuable founder animals by assisted reproductive technologies. Here, we report a comparison of processing methods by measuring the motility of fresh and frozen-thawed rhesus monkey spermatozoa and fertility via intracytoplasmic spermatozoa injection (ICSI) of sibling oocytes. Washed spermatozoa were frozen in straws or in pellets using different cryoprotective media and processed post-thaw with or without a density gradient centrifugation step. Among the four study series, motility post-thaw was improved with density gradient centrifugation (17-24% versus 75%, P<0.01) achieving levels similar to fresh spermatozoa. Spermatozoa injected oocytes (total n=377) were co-cultured on BRL cells and observed for fertilization and development. With spermatozoa frozen in straws in liquid nitrogen vapors, the fertilization rate after ICSI was lower than with fresh spermatozoa (40-44% versus 77-86%, P<0.05), even with the Percoll-enriched fraction that exhibited robust motility. In contrast, somewhat slower freezing of spermatozoa in pellets on dry ice supported fertilization rates (73%) that were similar to the fresh counterpart. Developmental rates of fertilized eggs were similar in all experiments. A total of 106 embryo transfers has resulted in the first primate born after ICSI with F/T ejaculated spermatozoa plus 22 other infants to date. Additionally, a 3-4 h incubation after thawing improved the fertilization rate with spermatozoa from a male with poor post-thaw recovery of sperm motility. In conclusion, an acceptable fertilization rate after ICSI with motile, frozen-thawed primate spermatozoa was observed comparable to that obtained with fresh spermatozoa allowing small quantities of competent spermatozoa to be used with ICSI to facilitate propagation of desirable primate genotypes.

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PubMed2005

Challenges of primate embryonic stem cell research.

Embryonic stem (ES) cells hold great promise for treating degenerative diseases, including diabetes, Parkinson's, Alzheimer's, neural degeneration, and cardiomyopathies. This research is controversial to some because producing ES cells requires destroying embryos, which generally means human embryos. However, some of the surplus human embryos available from in vitro fertilization (IVF) clinics may have a high rate of genetic errors and therefore would be unsuitable for ES cell research. Although gross chromosome errors can readily be detected in ES cells, other anomalies such as mitochondrial DNA defects may have gone unrecognized. An insurmountable problem is that there are no human ES cells derived from in vivo-produced embryos to provide normal comparative data. In contrast, some monkey ES cell lines have been produced using in vivo-generated, normal embryos obtained from fertile animals; these can represent a "gold standard" for primate ES cells. In this review, we argue a need for strong research programs using rhesus monkey ES cells, conducted in parallel with studies on human ES and adult stem cells, to derive the maximum information about the biology of normal stem cells and to produce technical protocols for their directed differentiation into safe and functional replacement cells, tissues, and organs. In contrast, ES cell research using only human cell lines is likely to be incomplete, which could hinder research progress, and delay or diminish the effective application of ES cell technology to the treatment of human diseases.

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PubMed2004

Assisted reproductive technologies in rhesus macaques.

The assisted reproductive technologies (ARTs) have been used in the production of rhesus monkey offspring at the Oregon National Primate Research Center (ONPRC) and that experience is summarized here. Additionally these technologies serve as a source of oocytes/embryos for monozygotic twinning, embryonic stem (ES) cell derivation and cloning. High fertilization efficiencies were realized with conventional insemination or following the use of intracytoplasmic sperm injection (ICSI) and approximately 50% of the resulting embryos grew in vitro to blastocysts. Both fresh and frozen sperm were employed in fertilization by ICSI and the resulting embryos could be low temperature stored for subsequent thawing and transfer when a synchronized recipient female was available or after shipment to another facility. Following the transfer of up to 3 embryos, an overall pregnancy rate of 30% was achieved with increasing rates dependent upon the number of embryos transferred. Singleton pregnancy outcomes following the transfer of ART produced embryos were similar to those observed in a control group of animals in the timed mated breeding colony at ONPRC. ICSI produced embryos were used in efforts to create monozygotic twins by blastomere separation or blastocyst splitting. While pregnancies were achieved following the transfer of demi-embryos, only one was a twin and it was lost to spontaneous abortion. ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells. These pluripotent cells hold potential for cell based therapies and we consider the monkey an important translational model in which to evaluate safety, efficacy and feasibility of regenerative medicine approaches based on the transplantation of stem cell-derived progeny. Finally, efforts to produce genetically-identical monkeys by nuclear transfer have been briefly summarized.

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PubMed2004

Derivation and characterization of monkey embryonic stem cells.

Embryonic stem (ES) cell based therapy carries great potential in the treatment of neurodegenerative diseases. However, before clinical application is realized, the safety, efficacy and feasibility of this therapeutic approach must be established in animal models. The rhesus macaque is physiologically and phylogenetically similar to the human, and therefore, is a clinically relevant animal model for biomedical research, especially that focused on neurodegenerative conditions. Undifferentiated monkey ES cells can be maintained in a pluripotent state for many passages, as characterized by a collective repertoire of markers representing embryonic cell surface molecules, enzymes and transcriptional factors. They can also be differentiated into lineage-specific phenotypes of all three embryonic germ layers by epigenetic protocols. For cell-based therapy, however, the quality of ES cells and their progeny must be ensured during the process of ES cell propagation and differentiation. While only a limited number of primate ES cell lines have been studied, it is likely that substantial inter-line variability exists. This implies that diverse ES cell lines may differ in developmental stages, lineage commitment, karyotypic normalcy, gene expression, or differentiation potential. These variables, inherited genetically and/or induced epigenetically, carry obvious complications to therapeutic applications. Our laboratory has characterized and isolated rhesus monkey ES cell lines from in vitro produced blastocysts. All tested cell lines carry the potential to form pluripotent embryoid bodies and nestin-positive progenitor cells. These ES cell progeny can be differentiated into phenotypes representing the endodermal, mesodermal and ectodermal lineages. This review article describes the derivation of monkey ES cell lines, characterization of the undifferentiated phenotype, and their differentiation into lineage-specific, particularly neural, phenotypes. The promises and limitations of primate ES cell-based therapy are also discussed.

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PubMed2004

Directed differentiation of rhesus monkey ES cells into pancreatic cell phenotypes.

Embryonic stem cells (ES) can self-replicate and differentiate into all cell types including insulin-producing, beta-like cells and could, therefore, be used to treat diabetes mellitus. To date, results of stem cell differentiation into beta cells have been debated, largely due to difficulties in defining the identity of a beta cell. We have recently differentiated non-human primate (rhesus) embryonic stem (rES) cell lines into insulin producing, beta-like cells with the beta cell growth factor, Exendin-4 and using C-peptide as a phenotype marker. Cell development was characterized at each stage by gene and protein expression. Insulin, NKX6.1 and glucagon mRNA were expressed in stage 4 cells but not in early undifferentiated cells. We concluded that rES cells could be differentiated ex vivo to insulin producing cells. These differentiated rES cells could be used to develop a non-human primate model for evaluating cell therapy to treat diabetes. To facilitate the identification of beta-like cells and to track the cells post-transplantation, we have developed a marker gene construct: fusing the human insulin promoter (HIP) to the green fluorescent protein (GFP) gene. This construct was transfected into stage 3 rES derived cells and subsequent GFP expression was identified in C-peptide positive cells, thereby substantiating endogenous insulin production by rES derived cells. Using this GFP detection system, we will enrich our population of insulin producing rES derived cells and track these cells post-transplantation in the non-human primate model.

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PubMed2004

Progress with nonhuman primate embryonic stem cells.

Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

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PubMed2004

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.

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PubMed2003

Oct-4 expression in pluripotent cells of the rhesus monkey.

The POU (Pit-Oct-Unc)-domain transcription factor, Oct-4, has become a useful marker of pluripotency in the mouse. It is found exclusively in mouse preimplantation-stage embryos after embryonic genome activation and is a characteristic of mouse embryonic stem (ES) cells, and its absence in knockout mice precludes inner cell mass (ICM) formation in blastocysts. Expression of Oct-4 has also been associated with pluripotency in primate cells. Here, we undertook a systematic study of Oct-4 expression in rhesus macaque preimplantation embryos produced by intracytoplasmic sperm injection and in ES cells before and after exposure to differentiating conditions in vitro. We also evaluated Oct-4 expression as a means of monitoring the extent of reprogramming following somatic cell nuclear transfer. Oct-4 was detected by reverse transcription-polymerase chain reaction and immunocytochemistry with a monoclonal antibody. Monkey pronuclear-stage zygotes and cleaving embryos up to the 8-cell stage showed no detectable Oct-4. Nuclear staining for Oct-4 first became obvious at the 16-cell stage, and a strong signal was observed in morula and compact morula stages. Both ICM and trophectodermal cell nuclei of monkey early blastocysts were positive for Oct-4. However, the signal was diminished in trophectodermal cells of expanded blastocysts, whereas expression remained high in ICM nuclei. Similar to the mouse, hatched monkey blastocysts showed strong Oct-4 expression in the ICM, with no detectable signal in the trophectoderm. Undifferentiated monkey ES cells derived from the ICM of in vitro-produced blastocysts expressed Oct-4, consistent with their pluripotent nature, whereas ES cell differentiation was associated with signal loss. Therefore, Oct-4 expression in the monkey, as in the mouse, provides a useful marker for pluripotency after activation of the embryonic genome. Finally, the observed lack or abnormal expression of Oct-4 in monkey nuclear transfer embryos suggests inadequate nuclear reprogramming.

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PubMed2003

The golden anniversary of cloning: a celebratory essay.

May 2002 marked the golden anniversary of the first cloned tadpoles. We celebrate this anniversary, as nuclear transplantation of frog cells into enucleated eggs became the prototype for cloning insects, fish, and mammals. We briefly review the salient results from amphibian cloning. Extension of these studies to mammalian species led to cloning adult cells, important advances in understanding nuclear reprogramming, and the construction of transgenic clones for biomedical applications. In addition, murine cloning clarified two problems unresolved in frog cloning: the unequivocal demonstration that nuclei of fully differentiated cells can direct the formation of fertile adults, and that abnormal expression of genes was responsible for the endoderm and neural syndromes in Rana clones.

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PubMed2002

Differentiation of monkey embryonic stem cells into neural lineages.

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

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PubMed2002

Rhesus monkey embryos produced by nuclear transfer from embryonic blastomeres or somatic cells.

Production of genetically identical nonhuman primates would reduce the number of animals required for biomedical research and dramatically impact studies pertaining to immune system function, such as development of the human-immunodeficiency-virus vaccine. Our long-term goal is to develop robust somatic cell cloning and/or twinning protocols in the rhesus macaque. The objective of this study was to determine the developmental competence of nuclear transfer (NT) embryos derived from embryonic blastomeres (embryonic cell NT) or fetal fibroblasts (somatic cell NT) as a first step in the production of rhesus monkeys by somatic cell cloning. Development of cleaved embryos up to the 8-cell stage was similar among embryonic and somatic cell NT embryos and comparable to controls created by intracytoplasmic sperm injection (ICSI; mean +/- SEM, 81 +/- 5%, 88 +/- 7%, and 87 +/- 4%, respectively). However, significantly lower rates of development to the blastocyst stage were observed with somatic cell NT embryos (1%) in contrast to embryonic cell NT (34 +/- 15%) or ICSI control embryos (46 +/- 6%). Development of somatic cell NT embryos was not markedly affected by donor cell treatment, timing of activation, or chemical activation protocol. Transfer of embryonic, but not of somatic cell NT embryos, into recipients resulted in term pregnancy. Future efforts will focus on optimizing the production of somatic cell NT embryos that develop in high efficiency to the blastocyst stage in vitro.

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