C. Chang I. Lee

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.

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.

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.

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.

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.

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 μCi/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 μCi/mL and rhMSCs radiolabeled with 10 μCi/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 μCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50 × 104 CD34+ cells (1.1 pCi/cell) and 6.25 × 103 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.

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.

Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells

Therapeutic angiogenesis is a promising strategy to promote the formation of new or collateral vessels for tissue regeneration and repair. Since changes in tissue oxygen concentrations are known to stimulate numerous cell functions, these studies have focused on the oxygen microenvironment and its role on the angiogenic potential of endothelial cells. We analyzed the proangiogenic potential of human endothelial colony-forming cells (hECFCs), a highly proliferative population of circulating endothelial progenitor cells, and compared outcomes to human dermal microvascular cells (HMVECs) under oxygen tensions ranging from 1% to 21% O2, representative of ischemic or healthy tissues and standard culture conditions. Compared to HMVECs, hECFCs (1) exhibited significantly greater proliferation in both ischemic conditions and ambient air; (2) demonstrated increased migration compared to HMVECs when exposed to chemotactic gradients in reduced oxygen; and (3) exhibited comparable or superior proangiogenic potential in reduced oxygen conditions when assessed using a vessel-forming assay. These data demonstrate that the angiogenic potential of both endothelial populations is influenced by the local oxygen microenvironment. However, hECFCs exhibit a robust angiogenic potential in oxygen conditions representative of physiologic, ischemic, or ambient air conditions, and these findings suggest that hECFCs may be a superior cell source for use in cell-based approaches for the neovascularization of ischemic or engineered tissues.

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.

Effect of age on the frequency, cell cycle, and lineage maturation of rhesus monkey (Macaca mulatta) CD34+ and hematopoietic progenitor cells

The effects of maturation and aging on hematopoietic progenitor cells, blood and bone marrow from second- and third-trimester fetal, newborn, infant, adult, and aged rhesus monkeys (Macaca mulatta) were analyzed. CD34+ cells were immunoselected and stained with propidium iodide for cell cycle analysis. Blood and bone marrow mononuclear cells were plated in methylcellulose, and erythroid and myeloid progenitors were grown and counted. A higher frequency of circulating CD34+CD38− and CD34+DR− cells was observed in second-trimester fetuses compared with the other age groups. The frequency of bone marrow CD34+CD38− and CD34+DR− cells declined in adult and aged animals when compared with the younger age groups. Cell-cycle analysis showed 4.5% second-trimester fetal bone marrow CD34+ cells entering the G2/M phase, compared with 1.7% CD34+ cells in aged animals. More than 95% of circulating CD34+ cells remained quiescent for most age groups, except for second-trimester fetuses. Adult marrow myeloid progenitors were found in a lower quantity when compared with third-trimester fetuses, whereas erythroid progenitors were greatest in early-gestation fetuses and adults. The results of these studies suggest that 1) the greatest quantity of CD34+CD38− and CD34+DR− cells was found in fetal and infant bone marrow, 2) the frequency of cycling CD34+ cells declines with maturation and aging, and 3) an age-dependent difference in lineage commitment occurs.