Special feature on stem cells: current research and future prospects

오일환,Evan Y Snyder 생화학분자생물학회 2013 Experimental and molecular medicine Vol.45 No.s

Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed

Kim, Hyoung-Tai,Kim, Il-Sun,Lee, Il-Shin,Lee, Jean-Pyo,Snyder, Evan Y.,In Park, Kook Elsevier 2006 Experimental neurology Vol.199 No.1

<P><B>Abstract</B></P><P>Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and γ-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.</P>

Implications and limitations of cellular reprogramming for psychiatric drug development

Brian TD Tobe,Michael G Brandel,Jeffrey S Nye,Evan Y Snyder 생화학분자생물학회 2013 Experimental and molecular medicine Vol.45 No.s

Human-induced pluripotent stem cells (hiPSCs) derived from somatic cells of patients have opened possibilities for in vitro modeling of the physiology of neural (and other) cells in psychiatric disease states. Issues in early stages of technology development include (1) establishing a library of cells from adequately phenotyped patients, (2) streamlining laborious, costly hiPSC derivation and characterization, (3) assessing whether mutations or other alterations introduced by reprogramming confound interpretation, (4) developing efficient differentiation strategies to relevant cell types, (5) identifying discernible cellular phenotypes meaningful for cyclic, stress induced or relapsing–remitting diseases, (6) converting phenotypes to screening assays suitable for genome-wide mechanistic studies or large collection compound testing and (7) controlling for variability in relation to disease specificity amidst low sample numbers. Coordination of material for reprogramming from patients well-characterized clinically, genetically and with neuroimaging are beginning, and initial studies have begun to identify cellular phenotypes. Finally, several psychiatric drugs have been found to alter reprogramming efficiency in vitro, suggesting further complexity in applying hiPSCs to psychiatric diseases or that some drugs influence neural differentiation moreso than generally recognized. Despite these challenges, studies utilizing hiPSCs may eventually serve to fill essential niches in the translational pipeline for the discovery of new therapeutics.

The dynamics of long-term transgene expression in engrafted neural stem cells

Lee, Jean-Pyo,Tsai, David J.,In Park, Kook,Harvey, Alan R.,Snyder, Evan Y. Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of comparative neurology Vol.515 No.1

<P>To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages. J. Comp. Neurol. 515:83–92, 2009. © 2009 Wiley-Liss, Inc.</P>

Neural stem cells may be uniquely suited for combined gene therapy and cell replacement: Evidence from engraftment of Neurotrophin-3-expressing stem cells in hypoxic–ischemic brain injury

Park, Kook In,Himes, B. Timothy,Stieg, Philip E.,Tessler, Alan,Fischer, Itzhak,Snyder, Evan Y. Elsevier 2006 Experimental neurology Vol.199 No.1

<P><B>Abstract</B></P><P>Previously, we reported that, when clonal neural stem cells (NSCs) were transplanted into brains of postnatal mice subjected to unilateral hypoxic–ischemic (HI) injury (optimally 3–7 days following infarction), donor-derived cells homed preferentially (from even distant locations) to and integrated extensively within the large ischemic areas that spanned the hemisphere. A subpopulation of NSCs and host cells, particularly in the penumbra, “shifted” their differentiation towards neurons and oligodendrocytes, the cell types typically damaged following asphyxia and least likely to regenerate spontaneously and in sufficient quantity in the “post-developmental” CNS. That no neurons and few oligodendrocytes were generated from the NSCs in intact postnatal cortex suggested that novel signals are transiently elaborated following HI to which NSCs might respond. The proportion of “replacement” neurons was ∼5%. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation during development and perhaps following injury. We demonstrated that NSCs express functional TrkC receptors. Furthermore, the donor cells continued to express a foreign reporter transgene robustly within the damaged brain. Therefore, it appeared feasible that neuronal differentiation of exogenous NSCs (as well as endogenous progenitors) might be enhanced if donor NSCs were engineered prior to transplantation to (over)express a bioactive gene such as NT-3. A subclone of NSCs transduced with a retrovirus encoding NT-3 (yielding >90% neurons in vitro) was implanted into unilaterally asphyxiated postnatal day 7 mouse brain (emulating one of the common causes of cerebral palsy). The subclone expressed NT-3 efficiently in vivo. The proportion of NSC-derived neurons increased to ∼20% in the infarction cavity and >80% in the penumbra. The neurons variously differentiated further into cholinergic, GABAergic, or glutamatergic subtypes, appropriate to the cortex. Donor-derived glia were rare, and astroglial scarring was blunted. NT-3 likely functioned not only on donor cells in an autocrine/paracrine fashion but also on host cells to enhance neuronal differentiation of both. Taken together, these observations suggest (1) the feasibility of taking a fundamental biological response to injury and augmenting it for repair purposes and (2) the potential use of migratory NSCs in some degenerative conditions for simultaneous combined gene therapy and cell replacement during the same procedure in the same recipient using the same cell (a unique property of cells with stem-like attributes).</P>

Acute injury directs the migration, proliferation, and differentiation of solid organ stem cells: Evidence from the effect of hypoxia–ischemia in the CNS on clonal “reporter” neural stem cells

In Park, Kook,Hack, Michael A.,Ourednik, Jitka,Yandava, Booma,Flax, Jonathan D.,Stieg, Philip E.,Gullans, Stephen,Jensen, Francis E.,Sidman, Richard L.,Ourednik, Vaclav,Snyder, Evan Y. Elsevier 2006 Experimental neurology Vol.199 No.1

<P><B>Abstract</B></P><P>Clonal neural cells with stem-like features integrate appropriately into the developing and degenerating central and peripheral nervous system throughout the neuraxis. In response to hypoxic–ischemic (HI) injury, previously engrafted, integrated, and quiescent clonal neural stem cells (NSCs) transiently re-enter the cell cycle, migrate preferentially to the site of ischemia, and differentiate into neurons and oligodendrocytes, the neural cell types typically lost following HI brain injury. They also replenish the supply of immature uncommitted resident stem/progenitor cells. Although they yield astrocytes, scarring is inhibited. These responses appear to occur most robustly within a 3–7 day “window” following HI during which signals are elaborated that upregulate genetic programs within the NSC that mediate proliferation, migration, survival, and differentiation, most of which appear to be terminated once the “window closes” and the chronic phase ensues, sending the NSCs into a quiescent state. These insights derived from using the stem cell in a novel role – as a “reporter” cell – to both track and probe the activity of endogenous stem cells as well as to “interrogate” and “report” the genes differentially induced by the acutely vs. chronically injured milieu. NSCs may be capable of the replacement of cells, genes, and non-diffusible factors in both a widespread or more circumscribed manner (depending on the therapeutic demands of the clinical situation). They may be uniquely responsive to some types of neurodegenerative conditions. We submit that these various capabilities are simply the normal expression of the basic homeostasis-preserving biologic properties and attributes of a stem cell which, if used rationally and in concert with this biology, may be exploited for therapeutic ends.</P>

Expression profile of an operationally-defined neural stem cell clone

Parker, Mark A.,Anderson, Julia K.,Corliss, Deborah A.,Abraria, Victoria E.,Sidman, Richard L.,Park, Kook In,Teng, Yang D.,Cotanche, Douglas A.,Snyder, Evan Y. Elsevier 2005 Experimental neurology Vol.194 No.2

<P><B>Abstract</B></P><P>Neural stem cells (NSCs) are the most primordial and least committed cells of the nervous system, the cells that exist <I>before</I> regional specification develops. Because immunocytochemically-detectable markers that are sufficiently specific and sensitive to define an NSC have not yet been fully defined, we have taken the strong view that, to be termed a “stem cell” in the nervous system—in contrast to a “progenitor” or “precursor” (whose lineage commitment is further restricted)—a <I>single neuroectodermally-derived cell</I> must fulfill an operational definition that is essentially similar to that used in hematopoiesis. In other words, it must possess the following functional properties: (1) “Multipotency”, i.e., the ability to yield mature cells in all three fundamental neural lineages throughout the nervous system—neurons (of all subtypes), astrocytes (of all types), oligodendrocytes—in multiple regional and developmental contexts and in a region and developmental stage-appropriate manner. (2) The ability to populate a developing region and/or repopulate an ablated or degenerated region of the nervous system with appropriate cell types. (3) The ability to be serially transplanted. (4) “Self-renewal”, i.e., the ability to produce daughter cells (including new NSCs) with identical properties and potential. Having identified a murine neural cell clone that fulfills this strict operational definition—in contrast to other studies that used less rigorous or non-operational criteria for defining an NSC (e.g., the “neurosphere” assay)—we then examined, by comparing gene expression profiles, the relationship such a cell might have to (a) a <I>multipotent</I> somatic stem cell from another organ system (the hematopoietic stem cell [HSC]); (b) a <I>pluripotent</I> stem cell derived from the inner cell mass and hence without organ assignment (an embryonic stem cell); (c) neural cells isolated and maintained primarily as neurospheres but without having been subjected to the abovementioned operational screen (“CNS-derived neurospheres”). ESCs, HSCs, and operationally-defined NSCs—all of which have been identified not only by markers but by functional assays in their respective systems and whose state of differentiation could be synchronized—shared a large number of genes. Although, as expected, the most stem-like genes were expressed by ESCs, NSCs and HSCs shared a number of genes. CNS-derived neurospheres, on the other hand, expressed fewer “stem-like” genes held in common by the other operationally-defined stem cell populations. Rather they displayed a profile more consistent with differentiated neural cells. (Genes of neural identity were shared with the NSC clone.) Interestingly, when the operationally-defined NSC clone was cultured as a neurosphere (rather than in monolayer), its expression pattern shifted from a “stem-like” pattern towards a more “differentiated” one, suggesting that the neurosphere, without functional validation, may be a poor model for predicting stem cell attributes because it consists of heterogeneous populations of cells, only a small proportion of which are truly “stem-like”. Furthermore, when operational definitions are employed, a common set of stem-like genes does emerge across both embryonic and somatic stem cells of various organ systems, including the nervous system.</P>

Generating iPSCs: Translating Cell Reprogramming Science into Scalable and Robust Biomanufacturing Strategies

Silva, M.,Daheron, L.,Hurley, H.,Bure, K.,Barker, R.,Carr, Andrew J.,Williams, D.,Kim, H.W.,French, A.,Coffey, Pete J.,Cooper-White, Justin J.,Reeve, B.,Rao, M.,Snyder, Evan Y.,Ng, Kelvin S.,Mead, Ben Cell Press 2015 Cell stem cell Vol.16 No.1

Induced pluripotent stem cells (iPSCs) have the potential to transform drug discovery and healthcare in the 21<SUP>st</SUP> century. However, successful commercialization will require standardized manufacturing platforms. Here we highlight the need to define standardized practices for iPSC generation and processing and discuss current challenges to the robust manufacture of iPSC products.