Cory R. Nicholas

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

Molecular identity of human outer radial glia during cortical development.

Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex.

Interneurons from Embryonic Development to Cell-Based Therapy

Background Alterations in neural excitation and inhibition cause a number of neurologic and psychiatric disorders. In the cerebral cortex, excitation and inhibition are mediated by two cell types born in distinct areas of the embryo: excitatory projection neurons, which are generated in the developing cortex, and inhibitory interneurons, which are produced outside the cortex in the ventral forebrain. After migrating from their origins across the developing brain, young interneurons reach the cortex and differentiate into various inhibitory neuronal cell types. Roughly two-thirds of these young cells survive in the cortex to form the local inhibitory circuits that shape excitatory neuron activity. The embryologic programs that guide interneuron migration, survival, and circuit integration are also executed by these young neurons after their transplantation into the juvenile and adult nervous systems. These processes, realized in the developmentally and topographically distinct environment of the recipient, offer a unique opportunity for studying neurodevelopment and therapeutically modifying neural circuits. Transplanted interneurons for the study of neural development and the treatment of nervous system disorders. Precursors of inhibitory interneurons transplanted from the medial ganglionic eminence of the ventral embryonic forebrain into the juvenile or adult rodent cortex migrate from the graft site and become dispersed throughout the recipient tissue (shown as small red dots in the transplanted hemisphere in a cross section of the rodent brain, upper left). In the recipient, transplanted interneurons follow cell-intrinsic programs that normally regulate their survival and differentiation in the embryo. Interneurons in the host brain (small green dots) do not die as a result of the additional neurons; rather, transplantation increases the total interneuron population. Transplanted interneurons develop axonal and dendritic arbors (red cell magnified in foreground), synaptically integrate into neural circuits, and modify inhibitory signaling. Interneuron transplantation provides a method for studying neural circuit assembly and function and is a potential cell-based therapy for conditions such as epilepsy, Parkinson’s disease, schizophrenia, anxiety, and chronic pain. Advances In both neonatal and adult rodents, transplanted embryonic interneurons have been shown to migrate and survive in diverse neural structures, including the cerebral cortex and the spinal cord. Transplanted interneurons form elaborate processes in host tissues, receive synaptic inputs, and make inhibitory connections with host neurons, similar to what they do in their normal setting. Functionally, transplantation has been used to modify inhibitory signaling in the host brain and to induce reorganization of the cortex by creating new windows of neural circuit plasticity. Transplanted interneurons have been shown to modify disease phenotypes in several rodent models of neurologic and psychiatric disorders, including epilepsy, chronic pain, Parkinson’s disease, schizophrenia, and anxiety. Interneuron transplantation has also been used to explore how cell-intrinsic and environmental factors interact to govern cellular fate and circuit formation. To generate interneurons for possible clinical applications, researchers are developing in vitro culture systems for the derivation of interneurons from embryonic stem cells and induced pluripotent stem cells. These efforts have produced new interneurons that, like their endogenous counterparts, disperse and integrate in the recipient brain after transplantation. Outlook Cortical interneurons are a heterogeneous population, and little is known about how distinct subtypes of interneurons function in neural circuits. Thus far, transplantation studies have used donor pools containing large mixtures of interneurons. As the mechanisms underlying interneuron diversity become better understood, donor populations may be selected or produced to include only specific subtypes of cells. This will allow researchers to study the functional roles of different interneuron types and may permit the use of specific donor populations for different pathologies. It is unknown how transplanted interneurons modify disease phenotypes. While transplanted interneurons likely exert therapeutic effects by increasing neural inhibition, other mechanisms are also possible. By transplanting mutant cells, or cells engineered to respond to optogenetic or chemical stimulation, these mechanisms may be elucidated. Eventual clinical applications will require more subtle and detailed studies of the behavioral effects of interneuron transplantation. Interneurons Reach Far and Wide Interneurons in the brain have been garnering increasing attention. Southwell et al. (10.1126/science.1240622) review the development of this unique class of neurons. The cells migrate long distances during brain development. Transplantation of interneurons derived from embryonic stem cells is yielding insight into disease processes and may have therapeutic potential. For example, Parkinsons disease, epilepsy, certain psychiatric disorders, and even some sorts of chronic pain either involve interneurons or may respond to transplanted interneurons. Many neurologic and psychiatric disorders are marked by imbalances between neural excitation and inhibition. In the cerebral cortex, inhibition is mediated largely by GABAergic (γ-aminobutyric acid–secreting) interneurons, a cell type that originates in the embryonic ventral telencephalon and populates the cortex through long-distance tangential migration. Remarkably, when transplanted from embryos or in vitro culture preparations, immature interneurons disperse and integrate into host brain circuits, both in the cerebral cortex and in other regions of the central nervous system. These features make interneuron transplantation a powerful tool for the study of neurodevelopmental processes such as cell specification, cell death, and cortical plasticity. Moreover, interneuron transplantation provides a novel strategy for modifying neural circuits in rodent models of epilepsy, Parkinson’s disease, mood disorders, and chronic pain.

Transplantation directs oocyte maturation from embryonic stem cells and provides a therapeutic strategy for female infertility

Ten to 15% of couples are infertile, with the most common causes being linked to the production of few or no oocytes or sperm. Yet, our understanding of human germ cell development is poor, at least in part due to the inaccessibility of early stages to genetic and developmental studies. Embryonic stem cells (ESCs) provide an in vitro system to study oocyte development and potentially treat female infertility. However, most studies of ESC differentiation to oocytes have not documented fundamental properties of endogenous development, making it difficult to determine the physiologic relevance of differentiated germ cells. Here, we sought to establish fundamental parameters of oocyte development during ESC differentiation to explore suitability for basic developmental genetic applications using the mouse as a model prior to translating to the human system. We demonstrate a timeline of definitive germ cell differentiation from ESCs in vitro that initially parallels endogenous oocyte development in vivo by single-cell expression profiling and analysis of functional milestones including responsiveness to defined maturation media, shared genetic requirement of Dazl, and entry into meiosis. However, ESC-derived oocyte maturation ultimately fails in vitro. To overcome this obstacle, we transplant ESC-derived oocytes into an ovarian niche to direct their functional maturation and, thereby, present rigorous evidence of oocyte physiologic relevance and a potential therapeutic strategy for infertility.

Instructing an Embryonic Stem Cell-Derived Oocyte Fate: Lessons from Endogenous Oogenesis

Female reproductive potential is limited in the majority of species due to oocyte depletion. Because functional human oocytes are restricted in number and accessibility, a robust system to differentiate oocytes from stem cells would enable a thorough investigation of the genetic, epigenetic, and environmental factors affecting human oocyte development. Also, the differentiation of functional oocytes from stem cells may permit the success of human somatic cell nuclear transfer for reprogramming studies and for the production of patient-specific embryonic stem cells (ESCs). Thus, ESC-derived oocytes could ultimately help to restore fertility in women. Here, we review endogenous and ESC-derived oocyte development, and we discuss the potential and challenges for differentiating functional oocytes from ESCs.

Characterization of a Dazl-GFP germ cell-specific reporter

In this study, we characterized the promoter activity of a 1.7 kb sequence in the 5′ flanking region of the mouse Deleted in Azoospermia‐Like (Dazl) gene. We found the 1.7 kb sequence sufficient to drive robust germ cell‐specific expression of green fluorescent protein (GFP) in adult mouse testis and lower levels of expression in adult ovary and in fetal and newborn gonads of both sexes. This expression pattern was confirmed in two independently‐derived transgenic mouse lines. In adult testis, Dazl‐GFP exhibited a developmentally‐regulated, stage‐specific expression pattern during spermatogenesis. GFP was highly expressed in spermatocyte stages, with strongest expression in pachytene spermatocytes. Weaker expression was observed in round and elongating spermatids, as well as spermatogonial cells. In the fetal gonad, GFP transcript was detected by e12.5 in both sexes; however, GFP fluorescence was only detected during later embryonic stages. In addition, we produced mouse embryonic stem cell (ESC) lines harboring the Dazl‐GFP reporter and used this reporter to isolate putative germ cell populations derived from mouse ESCs following embryoid body differentiation and fluorescence activated cell sorting. genesis 47:74–84, 2009.

Regenerative medicine: Cell reprogramming gets direct

In a feat of biological wizardry, one type of differentiated cell has been directly converted into another, completely distinct type. Notably, the approach does not require a stem-cell intermediate stage.

Intact fetal ovarian cord formation promotes mouse oocyte survival and development

BackgroundFemale reproductive potential, or the ability to propagate life, is limited in mammals with the majority of oocytes lost before birth. In mice, surviving perinatal oocytes are enclosed in ovarian follicles for subsequent oocyte development and function in the adult. Before birth, fetal germ cells of both sexes develop in clusters, or germline cysts, in the undifferentiated gonad. Upon sex determination of the fetal gonad, germ cell cysts become organized into testicular or ovarian cord-like structures and begin to interact with gonadal somatic cells. Although germline cysts and testicular cords are required for spermatogenesis, the role of cyst and ovarian cord formation in mammalian oocyte development and female fertility has not been determined.ResultsHere, we examine whether intact fetal ovarian germ and somatic cell cord structures are required for oocyte development using mouse gonad re-aggregation and transplantation to disrupt gonadal organization. We observed that germ cells from disrupted female gonad prior to embryonic day e13.5 completed prophase I of meiosis but did not survive following transplantation. Furthermore, re-aggregated ovaries from e13.5 to e15.5 developed with a reduced number of oocytes. Oocyte loss occurred before follicle formation and was associated with an absence of ovarian cord structure and ovary disorganization. However, disrupted ovaries from e16.5 or later were resistant to the re-aggregation impairment and supported robust oocyte survival and development in follicles.ConclusionsThus, we demonstrate a critical window of oocyte development from e13.5 to e16.5 in the intact fetal mouse ovary, corresponding to the establishment of ovarian cord structure, which promotes oocyte interaction with neighboring ovarian somatic granulosa cells before birth and imparts oocytes with competence to survive and develop in follicles. Because germline cyst and ovarian cord structures are conserved in the human fetal ovary, the identification of genetic components and molecular mechanisms of pre-follicle stage germ and somatic cell structures may be important for understanding human female infertility. In addition, this work provides a foundation for development of a robust fetal ovarian niche and transplantation based system to direct stem cell-derived oocyte differentiation as a potential therapeutic strategy for the treatment of infertility.

Use of “MGE Enhancers” for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinsons disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6+ cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6+ cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP+ cells, while enhancer 1056 is active in Olig2+ cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments.

Secretagogin is Expressed by Developing Neocortical GABAergic Neurons in Humans but not Mice and Increases Neurite Arbor Size and Complexity

The neocortex of primates, including humans, contains more abundant and diverse inhibitory neurons compared with rodents, but the molecular foundations of these observations are unknown. Through integrative gene coexpression analysis, we determined a consensus transcriptional profile of GABAergic neurons in mid-gestation human neocortex. By comparing this profile to genes expressed in GABAergic neurons purified from neonatal mouse neocortex, we identified conserved and distinct aspects of gene expression in these cells between the species. We show here that the calcium-binding protein secretagogin (SCGN) is robustly expressed by neocortical GABAergic neurons derived from caudal ganglionic eminences (CGE) and lateral ganglionic eminences during human but not mouse brain development. Through electrophysiological and morphometric analyses, we examined the effects of SCGN expression on GABAergic neuron function and form. Forced expression of SCGN in CGE-derived mouse GABAergic neurons significantly increased total neurite length and arbor complexity following transplantation into mouse neocortex, revealing a molecular pathway that contributes to morphological differences in these cells between rodents and primates.