Daniel F. Jimenez

Fetal CD34+ cells in the maternal circulation and long-term microchimerism in rhesus monkeys (Macaca mulatta)

Background. Studies in humans have shown that during pregnancy fetal cells can enter the maternal circulation and persist for many years. While we have previously reported the presence of cell-free fetal DNA during pregnancy in rhesus monkeys, it is unknown whether cells circulate and persist long term in maternal tissues. In this study, we asked whether fetal CD34+ cells can be found in the maternal circulation and if male fetal cells persist in maternal tissues postdelivery. Methods. The presence of the Y chromosome in maternal blood and tissues was assessed using real-time PCR assays for the sex determining region Y (SRY) and testes specific protein Y (TSPY) genes. Analysis was done on CD34+ and CD34− cells isolated from maternal blood collected at select time points during gestation from gravid animals with male or female fetuses, and tissues were analyzed from nongravid animals that had previously delivered male offspring. Results. All animals with male fetuses tested positive for the Y chromosome in CD34+ cells (0–30 cells/50,000 genome equivalents). Y sequences were also found in one or more maternal tissues collected up to 3-years postdelivery (thyroid, heart, spleen, liver, pituitary, adrenals, skin, inguinal lymph nodes). Conclusion. These studies suggest transfer of fetal CD34+ cells during pregnancy and persistent fetal microchimerism in the rhesus model. Thus, rhesus monkeys can be used to further our understanding of fetal:maternal microchimerism and the role of fetal cells in maternal health and disease.

HIV-1-derived lentiviral vectors and fetal route of administration on transgene biodistribution and expression in rhesus monkeys

The gene transfer efficiency of lentiviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein (VSV-G) driven by the MND or CMV promoters and expressing the enhanced green fluorescent protein (EGFP) was investigated in fetal rhesus monkeys (Macaca mulatta) (N=21). Fetuses (50±10 days gestation; term 165±10 days) were injected under ultrasound guidance using an intraperitoneal (i.p.) or intrahepatic (i.h.) approach with a range of 1 × 107–2 × 108 infectious particles/fetus. Analysis of transgene biodistribution and expression was performed in multiple tissues at 3–7 months postgene delivery using quantitative techniques. Overall, results indicated the following: (1) i.p. gene transfer at 40 days gestation resulted in a more diffuse distribution of the vector compared to administration at 60 days gestation; (2) vector biodistribution was similar after administration by the i.p. or i.h. routes; and (3) gene expression analysis in transduced tissues showed the presence of mRNA transcripts that correlated with the level of gene transfer. These studies suggest that fetal gene transfer using the i.p. and i.h. routes results in prolonged transduction and expression of the transgene in multiple tissues.

Quantitative analysis of male fetal DNA in maternal serum of gravid rhesus monkeys (Macaca mulatta).

The isolation of human fetal DNA from the maternal circulation has provided a source of fetal material for prenatal diagnosis. The objective of this study was to investigate whether a similar pattern could be observed in the maternal circulation of male-bearing gravid rhesus monkeys. A real-time PCR TaqMan system for the rhesus Y-chromosome sex determining region was used to determine fetal sex and to quantify fetal DNA concentrations. Results in 14 healthy pregnancies indicated that fetal male DNA could be routinely detected in maternal serum by 50 d of gestation (late first trimester; term 165 ± 10 d). Fetal DNA concentrations increased with advancing gestation, reaching a mean of 341 genome equivalents/mL of serum (range 11–1570 copies/mL) in the last trimester of gestation, similar to findings in humans. The fetal DNA concentration corresponded to 2.7% of the total maternal serum DNA in the third trimester. Similar to findings in humans, male fetal DNA sequences were not detected postpartum (through 4 wk postpartum) or in animals with a previous history of delivering male offspring. These data indicate that fetal male DNA is present in the maternal circulation of gravid rhesus monkeys comparable to findings in humans and further support the use of this nonhuman primate species as a model to investigate fetomaternal cell trafficking and microchimerism.

Fetal gender determination in early first trimester pregnancies of rhesus monkeys (Macaca mulatta) by fluorescent PCR analysis of maternal serum

Abstract:  Non‐human primate fetal gender determination can be a powerful tool for research study design and colony management purposes. The recent discovery of the presence of fetal DNA in maternal serum has offered a new non‐invasive approach for identification of fetal gender. We present a rapid and simple method for the sexing of developing rhesus monkeys in the first trimester by polymerase chain reaction (PCR) analysis of maternal serum. Serum samples were obtained from 72 gravid rhesus monkeys during 20–32 days of gestation (term 165 ± 10 days). Fetal gender and the quantity of circulating fetal DNA were determined by real‐time PCR analysis of the rhesus Y‐chromosomal DNA sequences. The sensitivity for identifying a male fetus was 100% by 30 days gestation, and no false‐positive results were observed. This study demonstrates that fetal gender can be reliably determined in the early first trimester from maternal serum samples, a non‐invasive method for routine gender screening.

Age-related gene expression profiles of rhesus monkey bone marrow-derived mesenchymal stem cells

The objective of this study was to elucidate age‐related differences in gene expression profiles of rhesus monkey bone marrow‐derived mesenchymal stem cells (rhMSC) obtained from fetal, infant, and adult donors relevant to their growth and other properties. Although a high degree of similarity was observed in the rhMSC gene expression profiles when comparing the three age groups, significant differences were found that strongly parallel gene expression profiles of human MSC. In general, there was a trend towards increased abundance of transcripts associated with differentiation and growth arrest with increasing donor age. Conversely, transcripts involved in RNA processing and the negative regulation of gene expression showed a downward trend with increasing donor age. Overall, the observed gene expression profiles were found to be similar to observations that MSC from older individuals show diminished proliferative capacity. These data highlight the importance of use of non‐human primates to study the properties of stem and progenitor cells, and for future therapies. J. Cell. Biochem. 103: 1198–1210, 2008.

462. Busulfan Dose Escalation to Increase Gene Marking of Hematopoietic Stem Cells by Lentiviral Vectors in Infant Rhesus Monkeys

Top of pageAbstract In clinical allogeneic bone marrow transplantation (BMT), complete myeloablation with high-dose busulfan (16 mg/kg) is often used to |[ldquo]|make space|[rdquo]| for the graft in the recipient BM compartment. As this treatment has significant toxicity, we sought to establish a low-dose busulfan protocol for partial myeloablation in the context of a gene-modified autologous BMT. Also, for infants and young children, calculation of dosage based on body surface area (mg/m2) has been reported give more consistent circulating busulfan levels than dosages based on mass (kg). In this study we sought to identify an optimal busulfan dose that could result in efficient long-term gene marking with minimal toxicities. We performed a lentiviral gene-marking study in infant rhesus macaques using escalating doses of busulfan. BM (|[sim]|10 ml/kg) was collected for immunoselection, and followed by a single 2 hour i.v. infusion of busulfan. Monkeys were transplanted using busulfan at 0, 40, 80, 120, and 160 mg/m2. Peripheral blood (PB) samples were collected for pharmacokinetic analysis up to 4 hours post-infusion, and the area under the curve (AUC) was determined. Isolated CD34+ cells were cultured for 24 hours in X-Vivo 15 with 100 ng/ml each of SCF, Flt3-L, and TPO. CD34+ cells were then transduced overnight with 4|[times]|10e7 infectious particles/ml of Simian Immunodeficiency Virus (SIV)-derived lentiviral vector pseudotyped with VSV-G on fibronectin-coated plates and using 4 |[mu]|g/ml protamine sulfate. The SIV vector contains a neomycin gene with a mutation in the start codon that abolishes its expression, and can therefore serve as a non-expressed marker gene. Transduced cells were washed and reinfused i.v 48 hours after administration of busulfan. Animals were monitored for myelosuppression, toxicity, and immune function. Increasing dosages of busulfan resulted in increased AUC. Some variability in AUC at each dose level was observed, suggesting inter-individual variations in busulfan absorption and clearance. At busulfan doses of 120 and 160 mg/m2, neutrophil and platelet counts transiently declined and were dose-dependent. No lymphopenia was observed, consistent with busulfan being myeloablative, but not immunosuppressive. Blood chemistries and behavior were normal in all animals, and no abnormalities were observed. PB and BM samples collected monthly were analyzed by real-time PCR to quantify gene marking. Gene marking levels have been fairly constant over the first 4 months post-transplant, ranging from undetectable in animals receiving no busulfan up to 0.1 % gene-containing cells in animals with the highest busulfan AUC. Together, these preliminary data suggest that (1) increased gene marking can be achieved by escalating busulfan doses, (2) busulfan is safe at the sub-myeloablative doses studied, and (3) while the anticipated myelosuppressive effects of busulfan were observed, there has been no evidence of adverse findings, to date.

743. Lentiviral Vector Gene Transfer in Monkeys: In Vivo. Detection of Gene Expression Longitudinally Using MicroPET and Optical Imaging

Top of pageAbstract Fetal intraperitoneal (IP) administration of HIV-1-derived lentiviral vectors (HIV/VSV-G/CMV/EGFP; 107 infectious particles/fetus) have consistently resulted in high levels of transduction and gene expression postnatally and for up to three years post-gene transfer in the omentum, peritoneum, and diaphragm when assessed by quantitative PCR and whole tissue fluorescence. Our objective was to develop in vivo imaging techniques that would allow us to monitor gene expression noninvasively and over time. In one series of studies, we explored the potential for imaging gravid long-tailed macaques with fetuses that were administered the VSV-G pseudotyped HIV-1-derived lentiviral vector expressing a mutant herpes simplex virus type 1 thymidine kinase (HSV-1 sr39tk) and firefly luciferase under the control of the CMV promoter IP in the first trimester. Fetuses were monitored sonographically during gestation to assess growth and development. Twice during the second and third trimesters 300-400 |[mu]|Ci F18-FHBG was injected into the fetal circulation under direct ultrasound guidance in preparation for maternal scanning in 3-D mode using a microPET 4 (CTI Concorde Microsystems). Images were reconstructed using an iterative MAP reconstruction algorithm. All newborns were delivered at term by cesarean-section and raised in the nursery for postnatal studies. Blood samples were collected for complete blood counts and chemistry panels monthly thereafter, and no adverse effects have been detected, to date. Beginning at two months postnatal age, animals were administered 650 |[mu]|Ci F18-FHBG and biodistribution assessed by microPET. In vivo optical imaging for firefly luciferase expression was also performed after injection of D-Luciferin using a Xenogen IVIS Imaging System with Living Image Software analysis. Under all imaging conditions gene expression was observed in the abdominal cavity, and closely paralleled findings in our prior studies using whole tissue fluorescence. These investigations have shown that HSV-1 sr39tk and firefly luciferase can be used to detect transgene expression longitudinally in fetal and infant monkeys in vivo, and without evidence of adverse effects.