Dosing and Re-Administration of Lentiviral Vector for In Vivo Gene Therapy in Rhesus Monkeys and ADA-Deficient Mice.

Adenosine deaminase (ADA)-deficient mice and healthy rhesus monkeys were studied to determine the impact of age at treatment, vector dosage, dosing schedule, repeat administration, biodistribution, and immunogenicity after systemic delivery of lentiviral vectors (LVs). In Ada -/- mice, neonatal treatment resulted in broad vector marking across all tissues analyzed, whereas adult treatment resulted in marking restricted to the liver, spleen, and bone marrow. Intravenous administration to infant rhesus monkeys also resulted in dose-dependent marking in the liver, spleen, and bone marrow. Using an ELISA to monitor anti-vector antibody development, Ada -/- neonatal mice did not produce an antibody response, whereas Ada -/- adult mice produced a strong antibody response to vector administration. In mice and monkeys with repeat administration of LV, a strong anti-vector antibody response was shown in response to the second LV administration, which resulted in LV inactivation. Three separate doses administered to immune competent mice resulted in acute toxicity. Pegylation of the vesicular stomatitis virus G protein (VSV-G)-enveloped LVs showed a less robust anti-vector response but did not prevent the inactivation of the second LV administration. These studies identify important factors to consider related to age and timing of administration when implementing systemic delivery of LVs as a potential therapeutic agent.

Systemic and Persistent Muscle Gene Expression in Rhesus Monkeys with a Liver De-Targeted Adeno-Associated Virus Vector.

The liver is a major off-target organ in gene therapy approaches for cardiac and musculoskeletal disorders. Intravenous administration of most of the naturally occurring adeno-associated virus (AAV) strains invariably results in vector genome sequestration within the liver. In the current study, we compared the muscle tropism and transduction efficiency of a liver de-targeted AAV variant to AAV9 following systemic administration in newborn rhesus monkeys. In vivo bioluminescence imaging was performed to monitor transgene expression (firefly luciferase) post administration. Results indicated comparable and sustained levels of systemic firefly luciferase gene expression in skeletal muscle over a period of two years. Quantitation of vector biodistribution in harvested tissues post-administration revealed widespread recovery of vector genomes delivered by AAV9 but markedly decreased levels in major systemic organs from the AAV variant. These studies validate the translational potential and safety of liver de-targeted AAV strains for gene therapy of muscle-related diseases.

Transfer of Therapeutic Genes into Fetal Rhesus Monkeys Using Recombinant Adeno-Associated Type I Viral Vectors.

Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa-/- mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 1012 particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.

Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Anti-non-Gal-specific combination treatment with an anti-idiotypic Ab and an inhibitory small molecule mitigates the xenoantibody response.

BACKGROUND: B-cell depletion significantly extends survival of α-1,3-galactosyltranferase knockout (GTKO) porcine organs in pig-to-primate models. Our previous work demonstrated that the anti-non-Gal xenoantibody response is structurally restricted. Selective inhibition of xenoantigen/xenoantibody interactions could prolong xenograft survival while preserving B-cell-mediated immune surveillance. METHODS: The anti-idiotypic antibody, B4N190, was selected from a synthetic human phage display library after enrichment against a recombinant anti-non-Gal xenoantibody followed by functional testing in vitro. The inhibitory small molecule, JMS022, was selected from the NCI diversity set III using virtual screening based on predicted xenoantibody structure. Three rhesus monkeys were pre-treated with anti-non-Gal-specific single-chain anti-idiotypic antibody, B4N190. A total of five monkeys, including two untreated controls, were then immunized with GTKO porcine endothelial cells to initiate an anti-non-α-1,3-Gal (non-Gal) xenoantibody response. The efficacy of the inhibitory small molecule specific for anti-non-Gal xenoantibody, JMS022, was tested in vitro. RESULTS: After the combination of in vivo anti-id and in vitro small molecule treatments, IgM xenoantibody binding to GTKO cells was reduced to pre-immunization levels in two-thirds of animals; however, some xenoantibodies remained in the third animal. Furthermore, when treated with anti-id alone, all three experimental animals displayed a lower anti-non-Gal IgG xenoantibody response compared with controls. Treatment with anti-idiotypic antibody alone reduced IgM xenoantibody response intensity in only one of three monkeys injected with GTKO pig endothelial cells. In the one experimental animal, which displayed reduced IgM and IgG responses, select B-cell subsets were also reduced by anti-id therapy alone. Furthermore, natural antibody responses, including anti-laminin, anti-ssDNA, and anti-thyroglobulin antibodies were intact despite targeted depletion of anti-non-Gal xenoantibodies in vivo indicating that selective reduction of xenoantibodies can be accomplished without total B-cell depletion. CONCLUSIONS: This preliminary study demonstrates the strength of approaches designed to selectively inhibit anti-non-Gal xenoantibody. Both anti-non-Gal-specific anti-idiotypic antibody and small molecules can be used to selectively limit xenoantibody responses.

Radiolabeling human peripheral blood stem cells for positron emission tomography (PET) imaging in young rhesus monkeys.

These studies focused on a new radiolabeling technique with copper ((64)Cu) and zirconium ((89)Zr) for positron emission tomography (PET) imaging using a CD45 antibody. Synthesis of (64)Cu-CD45 and (89)Zr-CD45 immunoconjugates was performed and the evaluation of the potential toxicity of radiolabeling human peripheral blood stem cells (hPBSC) was assessed in vitro (viability, population doubling times, colony forming units). hPBSC viability was maintained as the dose of (64)Cu-TETA-CD45 increased from 0 (92%) to 160 µCi/mL (76%, p>0.05). Radiolabeling efficiency was not significantly increased with concentrations of (64)Cu-TETA-CD45 >20 µCi/mL (p>0.50). Toxicity affecting both growth and colony formation was observed with hPBSC radiolabeled with ≥40 µCi/mL (p<0.05). For (89)Zr, there were no significant differences in viability (p>0.05), and a trend towards increased radiolabeling efficiency was noted as the dose of (89)Zr-Df-CD45 increased, with a greater level of radiolabeling with 160 µCi/mL compared to 0-40 µCi/mL (p<0.05). A greater than 2,000 fold-increase in the level of (89)Zr-Df-CD45 labeling efficiency was observed when compared to (64)Cu-TETA-CD45. Similar to (64)Cu-TETA-CD45, toxicity was noted when hPBSC were radiolabeled with ≥40 µCi/mL (p<0.05) (growth, colony formation). Taken together, 20 µCi/mL resulted in the highest level of radiolabeling efficiency without altering cell function. Young rhesus monkeys that had been transplanted prenatally with 25×10(6) hPBSC expressing firefly luciferase were assessed with bioluminescence imaging (BLI), then 0.3 mCi of (89)Zr-Df-CD45, which showed the best radiolabeling efficiency, was injected intravenously for PET imaging. Results suggest that (89)Zr-Df-CD45 was able to identify engrafted hPBSC in the same locations identified by BLI, although the background was high.

Characterization of growth, glomerular number, and tubular proteins in the developing rhesus monkey kidney.

An essential step in the translation of cell-based therapies for kidney repair involves preclinical studies in relevant animal models. Regenerative therapies in children with congenital kidney disease may provide benefit, but limited quantitative data on normal development is available to aid in identifying efficient protocols for repair. Nonhuman primates share many developmental similarities with humans and provide an important translational model for understanding nephrogenesis and morphological changes across gestation. These studies assessed monkey kidney size and weight during development and utilized stereological methods to quantitate total number of glomeruli. Immunohistochemical methods were included to identify patterns of expression of tubular proteins including Aquaporin-1 (AQP1), AQP2, Calbindin, E-Cadherin, and Uromodulin. Results have shown that glomerular number increased linearly with kidney weight, from 1.1 × 10(3) in the late first trimester to 3.5 × 10(5) near term (P < 0.001). The ratio of glomeruli to body weight tripled from the late first to early second trimester then remained relatively unchanged. Only AQP1 was expressed in the proximal tubule and descending Loop of Henle. The ascending Loop of Henle was positive for AQP2, Calbindin, and Uromodulin; distal convoluted tubules stained for Calbindin only; and collecting tubules expressed AQP2 and E-Cadherin with occasional Calbindin-positive cells. These findings provide quantitative information on normal kidney ontogeny in rhesus monkeys and further support the importance of this model for human kidney development.

Tissue specificity of decellularized rhesus monkey kidney and lung scaffolds.

Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.

Nonmyeloablative conditioning regimen to increase engraftment of gene-modified hematopoietic stem cells in young rhesus monkeys.

Immune responses to transgene products may lead to rejection of transduced cells, limiting successful gene therapy for genetic diseases. While moderate dosages of chemotherapeutic agents such as busulfan may increase hematopoietic stem cells (HSC) engraftment, they are not immune suppressive and do not abrogate immune responses to transgene products. Studies focused on nonmyeloablative conditioning with busulfan ± fludarabine in a clinically relevant monkey model to induce immune suppression to allow cells expressing a foreign transgene product to persist. Bone marrow CD34(+) HSC were transduced in two equal fractions using simian immunodeficiency virus (SIV)-based lentiviral vectors carrying a nonexpressed DNA sequence tag (NoN) and the green fluorescent protein (GFP) reporter gene. Post-transplant there was no evidence of elimination of cells containing the potentially immunogenic GFP gene; several recipients had stable persistence of cells, and no differences were detected with fludarabine, which was rapidly cleared. Antibodies and cellular immune responses to GFP developed in recipients with the highest levels of GFP-marked cells, although these cells were not eliminated. These studies establish a clinically relevant pediatric primate model to assess the effects of conditioning regimens on the engraftment of transduced HSC and the immune responses to cells expressing a foreign gene product.

Radiolabeling and in vivo imaging of transplanted renal lineages differentiated from human embryonic stem cells in fetal rhesus monkeys.

PURPOSE: The goals of this study were to optimize radiolabeling of renal lineages differentiated from human embryonic stem (hES) cells and use noninvasive imaging (positron emission tomography (PET) and bioluminescence imaging (BLI)) to detect the cells in fetal monkeys post-transplant. PROCEDURES: hES cells expressing firefly luciferase (5 × 10(6)) were radiolabeled with the optimized concentration of 10 µCi/ml (64)Cu-PTSM then transplanted under ultrasound guidance into early second trimester fetal monkey kidneys. Fetuses were imaged in utero with PET and tissues collected for analysis 3 days post-transplant. Fetal kidneys were imaged ex vivo (PET and BLI) post-tissue harvest, and serial kidney sections were assessed by PCR for human-specific DNA sequences, fluorescent in situ hybridization (FISH) for human-specific centromere probes, and immunohistochemistry (IHC) to assess engrafted cells. RESULTS: Transplanted cells were readily imaged in vivo and identified at the site of injection; tissue analyses confirmed the imaging findings. Using a semi-quantitative method, one in approximately 650 cells in the kidney was shown to be of human origin by PCR and FISH. CONCLUSIONS: These studies suggest that hES cells differentiated toward renal lineages can be effectively radiolabeled, transplanted into fetal monkey kidneys under ultrasound guidance, monitored with PET post-transplant, and identified by PET, BLI, PCR, FISH, and IHC post-tissue harvest.

Ontogeny of the kidney and renal developmental markers in the rhesus monkey (Macaca mulatta).

Nonhuman primates share many developmental similarities with humans, thus they provide an important preclinical model for understanding the ontogeny of biomarkers of kidney development and assessing new cell-based therapies to treat human disease. To identify morphological and developmental changes in protein and RNA expression patterns during nephrogenesis, immunohistochemistry and quantitative real-time PCR were used to assess temporal and spatial expression of WT1, Pax2, Nestin, Synaptopodin, alpha-smooth muscle actin (α-SMA), CD31, vascular endothelial growth factor (VEGF), and Gremlin. Pax2 was expressed in the condensed mesenchyme surrounding the ureteric bud and in the early renal vesicle. WT1 and Nestin were diffusely expressed in the metanephric mesenchyme, and expression increased as the Pax2-positive condensed mesenchyme differentiated. The inner cleft of the tail of the S-shaped body contained the podocyte progenitors (visceral epithelium) that were shown to express Pax2, Nestin, and WT1 in the early second trimester. With maturation of the kidney, Pax2 expression diminished in these structures, but was retained in cells of the parietal epithelium, and as WT1 expression was upregulated. Mature podocytes expressing WT1, Nestin, and Synaptopodin were observed from the mid-third trimester through adulthood. The developing glomerulus was positive for α-SMA (vascular smooth muscle) and Gremlin (mesangial cells), CD31 (glomerular endothelium), and VEGF (endothelium), and showed loss of expression of these markers as glomerular maturation was completed. These data form the basis for understanding nephrogenesis in the rhesus monkey and will be useful in translational studies that focus on embryonic stem and other progenitor cell populations for renal tissue engineering and repair.

Clonal analysis and hierarchy of human bone marrow mesenchymal stem and progenitor cells.

OBJECTIVE: This study was performed to assess adult human bone marrow mesenchymal stem/progenitor cells at a single-cell level and to determine a hierarchy based on proliferative potential. MATERIALS AND METHODS: Adult bone marrow mesenchymal cells expressing the enhanced green fluorescent protein (EGFP) were sorted as single cells into 24-well plates, each well confirmed with single EGFP-positive cells by fluorescence microscopy, and counted every 3 days. Colonies derived from single cells were expanded then sorted and evaluated using established differentiation protocols for adipogenic, chondrogenic, and osteogenic lineages. Cells were further analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) (peroxisome proliferator-activated receptor[PPAR]-gamma2, LEP, LPL, LUM, COMP, BIG, RUNX2, IBSP, BGLAP) and immunocytochemistry (PPAR-gamma1/2, collagen II, bone sialoprotein II) specific for trilineage differentiation. RESULTS: Bone marrow mesenchymal cells were found to contain high proliferative potential (HPP) mesenchymal colony-forming cells (MCFC) (7%), low proliferative potential (LPP) MCFC (29%), mesenchymal cell clusters (MCC, 26%), and mature mesenchymal cells (MMC, 38%). All LPP-MCFC, MCC, and MMC colonies reached senescence at the end of the evaluation period. However, HPP-MCFC continued to grow, showed differentiation toward all three lineages, and demonstrated the capacity to give rise to secondary HPP-MCFC upon replating at a clonal level. CONCLUSION: These findings suggest that there is a low frequency of bone marrow-derived HPP-MCFC that can both self-renew at a single-cell level and differentiate toward multiple lineages of mesenchymal origin.

Long-term luciferase expression monitored by bioluminescence imaging after adeno-associated virus-mediated fetal gene delivery in rhesus monkeys (Macaca mulatta).

The safety and efficiency of fetal adeno-associated virus (AAV) gene delivery in rhesus monkeys and long-term monitoring of transgene expression by bioluminescence imaging (BLI) were evaluated. Early second-trimester fetal monkeys were administered AAV2/5, AAV2/9, or AAV2/10 vector supernatant preparations expressing firefly luciferase under the control of the cytomegalovirus promoter, using an intrathoracic (n = 6) or intramyocardial (n = 6) approach and established ultrasound-guided techniques. Postnatal BLI was performed monthly up to 6 months postnatal age (n = 12) and then every 3 months thereafter to monitor transgene expression up to 24 months postnatal age (27 months after gene transfer; n = 6). All AAV serotypes showed greater than 1.0 x 10(9) photons/sec at all time points evaluated with limited biodistribution to nontargeted anatomical sites. The highest levels of bioluminescence (photons per second) observed were noted with AAV2/9 and AAV2/10 when the three vector constructs were compared. To correlate in vivo findings at the tissue level, specimens were collected from selected animals and analyzed. Three-dimensional reconstruction showed that firefly luciferase expression was consistent with imaging and morphometric measures. These findings suggest that (1) high levels of AAV-mediated firefly luciferase expression can be found after fetal AAV gene transfer and without any evidence of adverse effects; (2) the intercostal muscles, myocardium, and muscular component of the diaphragm of developing fetuses are readily transduced with AAV2/5, AAV2/9, or AAV2/10; and (3) postnatal outcomes and long-term luciferase expression can be effectively monitored by BLI in young rhesus monkeys.

Characterization and culture of fetal rhesus monkey renal cortical cells.

The renal glomerulus is composed of endothelial and mesangial cells with podocytes contributing to glomerular filtration. Podocyte damage is associated with renal disorders, thus there is interest in these cells for regenerative medicine. These studies investigated the use of extracellular matrix (ECM) to grow third trimester fetal monkey renal cortical cells and to assess mature podocytes in culture. Immunohistochemistry provided a profile of podocyte differentiation with metanephric mesenchyme and developing podocytes nestin positive and synaptopodin negative, whereas mature podocytes were positive for both markers. Primary cell cultures devoid of mature podocytes were established on plastic and renal ECM. A cell population (nestin+/synatopodin-) cultured on renal ECM showed greater proliferative potential compared with plastic with limited podocytes developing in culture over time. Further investigation of individual components of ECM (laminin, fibronectin, collagen I, or collagen IV) indicated that collagen I supported the greatest proliferation similar to renal ECM, whereas a greater number of mature podocytes (nestin+/synaptopodin+) were observed on fibronectin. These results suggest that (1) culture of fetal monkey podocytes can be accomplished, (2) renal ECM and collagen I can support renal cortical cells in vitro, which may recapitulate the developing kidney in vivo, and (3) fibronectin can support podocyte differentiation in vitro.

Real-time bioluminescence imaging of macroencapsulated fibroblasts reveals allograft protection in rhesus monkeys (Macaca mulatta).

BACKGROUND: Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte device was shown to provide long-term immunoprotection of murine islets in a mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner. METHODS: Encapsulated cells were transplanted subcutaneously (n=2), or cells were injected without encapsulation (n=1) and outcomes compared. BLI was performed to monitor cell survival. RESULTS: The BLI signal from the encapsulated cells remained robust postinsertion and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days. CONCLUSIONS: These data demonstrate that TheraCyte encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys.

Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors.

The development of the metanephric kidney was studied immunohistochemically across gestation in monkeys to identify markers of cell specification, and to aid in developing experimental paradigms for renal precursor differentiation from human embryonic stem cells (hESC). PAX2, an important kidney developmental marker, was expressed at the tips of the ureteric bud, in the surrounding condensing mesenchyme, and in the renal vesicle. Vimentin, a mesenchymal and renal marker, was strongly expressed in the metanephric blastema then found to be limited to the glomerulus and interstitial cells of the medulla and cortex. A model of gene expression based on human and nonhuman primate renal ontogeny was developed and incorporated into studies of hESC differentiation. Spontaneous hESC differentiation revealed markers of metanephric mesenchyme (OSR1, PAX2, SIX2, WT1) that increased over time, followed by upregulation of kidney precursor markers (EYA1, LIM1, CD24). Directed hESC differentiation was also evaluated with the addition of retinoic acid, Activin-A, and BMP-4 or BMP-7, and using different culture substrate conditions. Of the culture substrates studied, gelatin most closely recapitulated the anticipated directed developmental pattern of renal gene expression. No differences were found when BMP-4 and BMP-7 were compared with baseline conditions. PAX2 and Vimentin immunoreactivity in differentiating hESC was also similar to the renal precursor patterns reported for human fetal kidneys and findings described in rhesus monkeys. The results of these studies are as follows: (1) provide additional data to support that rhesus monkey kidney development parallels that of humans, and (2) provide a useful model for hESC directed differentiation towards renal precursors.

Radiolabeling rhesus monkey CD34+ hematopoietic and mesenchymal stem cells with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for microPET imaging.

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34+ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34+ hematopoietic cells were radiolabeled with 0 to 40 microCi/mL 64Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34+ cells radiolabeled with 20 microCi/mL and rhMSCs radiolabeled with 10 microCi/mL 64Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34+ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 microCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50x10(4) CD34+ cells (1.1 pCi/cell) and 6.25x10(3) rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34+ hematopoietic cells and rhMSCs can be safely radiolabeled with 64Cu-PTSM without adverse cellular effects.

Growth, differentiation, and biochemical signatures of rhesus monkey mesenchymal stem cells.

The goal of this study was to compare the growth and differentiation potential of rhesus monkey mesenchymal stem cells (rhMSCs) from different age groups (fetal, newborn, infant, juvenile), and to use confocal micro-Raman spectroscopy to assess the intrinsic biomolecular profiles of individual rhMSCs. Results indicated that fetal cells had significantly shorter population doubling times during the log growth phase (23.3 +/- 1.3 h) and greater population doubling times (66.5 +/- 6.5) when compared to other age groups (newborn 51.9 +/- 2.3, infant 38.2 +/- 3.1, juvenile 40.7 +/- 4.1). Fetal rhMSCs also differentiated toward osteogenic and adipogenic lineages at a faster rate when compared to cells from older animals. The Raman spectral analysis showed greater DNA and lower protein concentration in fetal compared to juvenile rhMSCs, although the spectra from different age groups shared many similar features. Additionally, principal component analysis (PCA), which is used to discriminate between rhMSCs, supported prior findings that suggested that cultured rhMSCs consist of a heterogeneous cell population. Although the growth potential of rhMSCs from the younger age groups was confirmed, further studies will be necessary to fully explore the potential usefulness of Raman micro-spectroscopy to characterize stem and progenitor cells such as rhMSCs.

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.

Fetal gene transfer using lentiviral vectors: in vivo detection of gene expression by microPET and optical imaging in fetal and infant monkeys.

Fetal intraperitoneal administration of human immunodeficiency virus (HIV)-l-derived lentiviral vectors (10(7) infectious particles/fetus) has consistently shown high levels of transduction and gene expression in the omentum, peritoneum, and diaphragm when assessed by polymerase chain reaction (PCR) and whole tissue fluorescence. In vivo imaging techniques were explored with early-gestation long-tailed macaques that were administered the vesicular stomatitis virus-glycoprotein (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 cytomegalovirus (CMV) promoter. Fetuses were monitored sonographically and twice during gestation 9-[4-[18F]Fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG) was injected into the fetal circulation under ultrasound guidance in preparation for microPET imaging. All newborns were delivered at term by cesarean section and raised in the nursery for postnatal studies. At 2 months postnatal age, animals were imaged and biodistribution was assessed. Optical imaging for firefly luciferase expression was also performed every 2 months postnatal age. Under all imaging conditions gene expression was observed in the abdominal region, and closely paralleled findings from prior studies based on whole tissue fluorescence. These investigations have shown that HSV-1-sr39tk and firefly luciferase can be used to safely detect transgene expression at multiple time points in fetal and infant monkeys in vivo and without evidence of adverse effects.