Juanito J. Meneses

Mutations of the homeobox genes Dlx-1 and Dlx-2 disrupt the striatal subventricular zone and differentiation of late born striatal neurons.

The striatum has a central role in many neurobiological processes, yet little is known about the molecular control of its development. Inroads to this subject have been made, due to the discovery of transcription factors, such as the Dlx genes, whose expression patterns suggest that they have a role in striatal development. We report that mice lacking both Dlx-1 and Dlx-2 have a time-dependent block in striatal differentiation. In these mutants, early born neurons migrate into a striatum-like region, which is enriched for markers of the striosome (patch) compartment. However, later born neurons accumulate within the proliferative zone. Several lines of evidence suggest that mutations in Dlx-1 and Dlx-2 produce abnormalities in the development of the striatal subventricular zone and in the differentiation of striatal matrix neurons.

Integrin α8β1 Is Critically Important for Epithelial–Mesenchymal Interactions during Kidney Morphogenesis

We present genetic evidence that integrins regulate epithelial–mesenchymal interactions during organogenesis. Mice with a mutation in the α8 gene do not express the integrin α8β1 and exhibit profound deficits in kidney morphogenesis. In wild-type animals, inductive interactions between the ureteric epithelium and metanephric mesenchyme are essential for kidney morphogenesis. In α8 mutant homozygotes, growth and branching of the ureteric bud and recruitment of mesenchymal cells into epithelial structures are defective. Consistent with these phenotypes, α8 expression is induced in mesenchymal cells upon contact with the ureter. Since none of its previously identified ligands appears likely to mediate the essential functions of α8β1 in kidney morphogenesis, we have used an α8β1–alkaline phosphatase chimera to localize novel ligand(s) in the growing ureter. The distribution of these ligand(s) makes them strong candidates for regulators of kidney morphogenesis.

An Olfactory Sensory Map Develops in the Absence of Normal Projection Neurons or GABAergic Interneurons

Olfactory sensory neurons expressing a given odorant receptor project to two topographically fixed glomeruli in the olfactory bulb. We have examined the contribution of different cell types in the olfactory bulb to the establishment of this topographic map. Mice with a homozygous deficiency in Tbr-1 lack most projection neurons, whereas mice with a homozygous deficiency in Dlx-1 and Dlx-2 lack most GABAergic interneurons. Mice bearing a P2-IRES-tau-lacZ allele and deficient in either Tbr-1 or Dlx-1/Dlx-2 reveal the convergence of axons to one medial and one lateral site at positions analogous to those observed in wild-type mice. These observations suggest that the establishment of a topographic map is not dependent upon cues provided by, or synapse formation with, the major neuronal cell types in the olfactory bulb.

Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia‐derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post‐transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming. STEM CELLS 2009;27:138–149

Cell fate and cell lineage in the endoderm of the presomite mouse embryo, studied with an intracellular tracer

The fate of the embryonic endoderm (generally called visceral embryonic endoderm) of midstreak to neural plate stages of the mouse embryo was studied by microinjecting horseradish peroxidase (HRP) into single axial endoderm cells in situ, and tracing the labeled descendants to early somite stages in vitro. Axial endoderm cells along the anterior fifth of the late streak/neural plate stage embryo contributed descendants either to the yolk sac endoderm or to the anterior intestinal portal. Cells of the exposed head process contributed to the trunk endoderm and notochord; neighboring endoderm cells contributed to the dorsal foregut. Contributions to the ventral foregut came from endoderm at, and anterior to, the distal tip of the younger, midstreak embryo (in which the head process was not yet exposed). Endoderm over the primitive streak contributed to the postsomite endoderm. We argue from these results and those in the literature that during gastrulation the axial embryonic endoderm is of mixed lineage: (1) an anterior population of cells is derived from primitive endoderm and contributes to the yolk sac endoderm; (2) a population at, and anterior to, the distal tip of the midstreak embryo, extending more anteriorly at late streak/neural plate stages, is presumed to emerge from primitive ectoderm at the beginning of gastrulation and contributes to the foregut and anterior intestinal portal; (3) the axial portion of the head process that begins to incorporate into the ventral surface at the late streak stage contributes to notochord and trunk endoderm. Cells or their descendants that were destined to die within 24 hr were evident at the midstreak stage. There was a linear trend in the incidence of cell death among labeled cells at the late streak/neural plate stages, ranging from 27% caudal to the node to 57% in the anterior fifth of the embryo. The surviving axial endoderm cells divided sufficiently fast to double the population in 24 hr.

Propagation and Maintenance of Undifferentiated Human Embryonic Stem Cells

Human embryonic stem (hES) cells, like other stem cells, have the capacity to self-renew without differentiation. Although hES cells can be differentiated to many different tissue types in vitro, clinical uses have not yet been realized from the study of hES cells. Anticipation that these cells would be immediately useful for creating models of human disease has not yet been fulfilled. However, because of their self-renewing and pluripotential nature, hES cells indeed hold unique promise for many areas of research and medicine. A major problem complicating developments in hES cell research is the difficulty of propagating and maintaining these cells in vitro without differentiation. This review addresses this problem and potential solutions in detail. In addition, the current state of research regarding the growth and maintenance of hES cells is summarized, along with basic protocols utilized by our laboratory for the successful propagation, characterization, and investigation of hES cells.

The Effect of Extreme Hypoxia and Glucose on the Repair of Potentially Lethal and Sublethal Radiation Damage by Mammalian Cells

Confluent monolayers of Chinese hamster overy cells repaired potentially lethal X-ray damage under aerobic conditions but did not repair such damage under extremely hypoxic conditions. However, add...

Targeted disruption of the Artemis murine counterpart results in SCID and defective V(D)J recombination that is partially corrected with bone marrow transplantation

Artemis is a mammalian protein, the absence of which results in SCID in Athabascan-speaking Native Americans (SCIDA). This novel protein has been implicated in DNA double-strand break repair and V(D)J recombination. We have cloned the Artemis murine counterpart, mArt, and generated a mouse with a targeted disruption of mArt. Artemis-deficient mice show a similar T−B− NK+ immunodeficiency phenotype, and carry a profound impairment in coding joint rearrangement, while retaining intact signal ends and close to normal signal joint formation. mArt−/− embryonic fibroblasts show increased sensitivity to ionizing radiation. Hemopoietic stem cell (HSC) transplantation using 500-5000 enriched congenic, but not allogeneic mismatched HSC corrected the T cell and partially corrected the B cell defect. Large numbers (40,000) of allogeneic mismatched HSC or pretreatment with 300 cGy of radiation overcame graft resistance, resulting in limited B cell engraftment. Our results suggest that the V(D)J and DNA repair defects seen in this mArt−/− mouse model are comparable to those in humans with Artemis deficiency, and that the recovery of immunity following HSC transplantation favors T rather than B cell reconstitution, consistent with what is seen in children with this form of SCID.

Expression of a mouse metallothionein-Escherichia coli β-galactosidase fusion gene (MT-βgal) in early mouse embryos☆

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli β-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolylβ-d-galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous β-galactosidase activity in 5–17% of DNA-injected embryos assayed at preimplantation stages after 16–24 h treatment with ZnSO4. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.

Characterization of six new human embryonic stem cell lines (HSF7, -8, -9, -10, -12, and -13) derived under minimal-animal component conditions.

Human embryonic stem cells (hESCs) provide a renewable source of a variety of cell types with the potential for use in both scientific research and clinical cell-based therapy. Several hESC lines have previously been isolated and characterized, however, the majority of these lines were generated in the presence of animal serum and animal-derived feeder cells. Therefore, the exposure of the hESC to animal products may have induced phenotypic and/or genomic changes in the hESC lines not characteristic of normal hESC. Moreover, those hESC lines exposed to animal components may not be used for therapeutic applications due to the risk of graft rejection and pathogenic transmission from animal sources. In this study, we characterized six new hESC lines derived from human blastocysts under minimal-animal component conditions and cultured with human fetal lung fibroblasts. The hESC lines retained the ability to self-renew, are karytopically normal, and express stage-specific embryonic antigen-3 (SSEA-3), SSEA-4, TRA-1-60, and TRA-1-81, but not SSEA-1, markers of pluripotent hESC. In addition, we show that telomerase activity decreased in each of the hESC lines following differentiation into embryoid bodies, albeit to different degrees. Finally, we demonstrate that the hESC lines are capable of differentiating into the three embryonic germ layers in vitro and form complex teratomas in vivo. This suggests that the hESC lines described here are valuable models for both future in vitro and in vivo studies, which may aid in the progression toward clinical-grade cell therapy.