Alice F. Tarantal

Establishment of a functional immune system has important implications for health and disease, yet questions remain regarding the mechanism, location, and timing of development of myeloid and lymphoid cell compartments. The goal of this study was to characterize the ontogeny of the myeloid‐lymphoid system in rhesus monkeys to enhance current knowledge of the developmental sequence of B‐cell (CD20, CD79), T‐cell (CD3, CD4, CD8, FoxP3), dendritic cell (CD205), and macrophage (CD68) lineages in the fetus and infant. Immunohistochemical assessments addressed the temporal and spatial expression of select phenotypic markers in the developing liver, thymus, spleen, lymph nodes, gut‐associated lymphoid tissue (GALT), and bone marrow with antibodies known to cross‐react with rhesus cells. CD3 was the earliest lymphoid marker identified in the first trimester thymus and, to a lesser extent, in the spleen. T‐cell markers were also expressed midgestation on cells of the liver, spleen, thymus, and in Peyer's patches of the small and large intestine, and where CCR5 expression was noted. A myeloid marker, CD68, was found on hepatic cells near blood islands in the late first trimester. B‐cell markers were observed mid‐second trimester in the liver, spleen, thymus, lymph nodes, bone marrow spaces, and occasionally in GALT. By the late third trimester and postnatally, secondary follicles with germinal centers were present in the thymus, spleen, and lymph nodes. These results suggest that immune ontogeny in monkeys is similar in temporal and anatomical sequence when compared to humans, providing important insights for translational studies. Anat Rec, 297:1392–1406, 2014.

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 × 10³ in the late first trimester to 3.5 × 10⁵ 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. Anat Rec, 296:1747–1757, 2013.

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 goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; “proof-of-principle”; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field.

Introduction: We have identified a novel hierarchy of human endothelial colony–forming cells (ECFCs) that are functionally defined by their proliferative and clonogenic potential and in vivo vessel-forming ability. The rhesus monkey provides an excellent model in which to examine the changes in circulating concentrations and functions of ECFCs since this nonhuman primate possesses a long lifespan and has been used extensively to model age-related processes that occur in humans. Results: Endothelial cells (ECs) derived from rhesus monkey ECFCs share a cell-surface phenotype similar to human cord blood ECFCs, rapidly form capillary-like structures in vitro, and form endothelial-lined vessels in vivo upon implantation in immunodeficient mice in an age-dependent manner. Of interest, although ECFCs from the oldest monkeys formed capillary-like structures in vitro, the cells failed to form inosculating vessels when implanted in vivo and displayed a deficiency in cytoplasmic vacuolation in vitro; a critical first step in vasculogenesis. Discussion: Utilizing previously established clonogenic assays for defining different subpopulations of human ECFCs, we have shown that a hierarchy of ECFCs, identical to human cells, can be isolated from the peripheral blood of rhesus monkeys, and that the frequency of the circulating cells varies with age. These studies establish the rhesus monkey as an important preclinical model for evaluating the role and function of circulating ECFCs in vascular homeostasis and aging. Methods: Peripheral blood samples were collected from 40 healthy rhesus monkeys from birth to 24 years of age for ECFC analysis including immunophenotyping, clonogenic assays, and in vivo vessel formation.

New therapies for severely damaged kidneys are needed due to limited regenerative capacity and organ donor shortages. The goal of this study was to repopulate decellularized kidney sections in vitro and to determine the impact of donor age on recellularization. This was addressed by generating decellularized kidney scaffolds from fetal, juvenile, and adult rhesus monkey kidney sections using a procedure that removes cellular components while preserving the structural and functional properties of the native extracellular matrix (ECM). Kidney scaffolds were recellularized using explants from different age groups (fetal, juvenile, adult) and fetal renal cell fractions. Results showed vimentin+ cytokeratin+ calbindin+ cell infiltration and organization around the scaffold ECM. The extent of cellular repopulation was greatest with scaffolds from the youngest donors, and with seeding of mixed fetal renal aggregates that formed tubular structures within the kidney scaffolds. These findings suggest that decellularized kidney sections from different age groups can be effectively repopulated with donor cells and the age of the donor is a critical factor in repopulation efficiency.

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.

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. Anat Rec, 2010.