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.

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 to 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: (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.

Fetal gene transfer was studied using intrapulmonary and intramyocardial transfer of SIN HIV-1-derived lentiviral vectors expressing EGFP in rhesus monkeys. Fetuses were monitored sonographically during gestation and tissue analyses performed at term or 3 months postnatal age. Animals remained healthy during the study period as evidenced by normal growth, development, hematology, clinical chemistry, echocardiography, and pulmonary function tests. Strong pulmonary fluorescence was observed postnatally after fetal intrapulmonary delivery of lenti-CMV, but not lenti-SP-C, and compared to nontransferred controls. High EGFP copy numbers were found by quantitative PCR with both vector constructs in lung lobes (

The goal of this study was to compare the growth and differentiation potential of fetal and adult rhesus monkey (Macaca mulatta) mesenchymal stem cells (rhMSCs). rhMSCs were obtained from healthy early third-trimester fetal (n = 3) and adult (n = 3) rhesus monkey bone marrow. Fetal rhMSCs were plated at 10, 50, 100, or 1,000 cells/cm(2) in medium containing 10% or 20% infant monkey serum (IMS) or fetal bovine serum (FBS). Fetal rhMSCs grown at 1,000 cells/cm(2) in 20% FBS showed faster growth rates and differentiation toward adipogenic, chondrogenic, and osteogenic lineages when compared to other culture conditions and to adult cells (p < 0.05). Fetal rhMSC showed higher population doubling times (11.3 +/- 0.5) when compared to adult cells (7.3 +/- 0.8) during the first three passages. Adult rhMSC did not grow beyond the third passage under all culture conditions, including those supplemented with insulin-like growth factor (IGF)-I, IGF-II, platelet-derived growth factor (PDGF), and fibroblast growth factor-2 (FGF-2). After the third passage, adult rhMSC cultures were observed with large syncytia and with evidence of apoptosis. Cells obtained from these cultures tested positive for simian foamy virus (SFV) by PCR, RT-PCR, and immunofluorescent assay. Adult rhMSCs cultured with 10 microM tenofovir, an antiviral agent, showed normal growth and differentiation for over 20 population doublings. These findings suggest that: (1) fetal rhMSCs possess greater self-renewal and differentiation potential when compared to adult cells; and (2) SFV can inhibit proliferation of adult rhMSCs in culture, whereas the addition of tenofovir can successfully suppress SFV replication in vitro and result in resumed growth.

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. Results-Bone marrow mesenchymal cells were found to contain high proliferative potentialmesenchymal colony-forming cells (HPP-MCFC, 7%), low proliferative potential-mesenchymal colony-forming cells (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. Methods-AdultConclusion-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.