Joseph J. Kim

Neurons in the Basal Forebrain Project to the Cortex in a Complex Topographic Organization that Reflects Corticocortical Connectivity Patterns: An Experimental Study Based on Retrograde Tracing and 3D Reconstruction

The most prominent feature of the Basal Forebrain (BF) is the collection of large cortically projecting neurons (basal nucleus of Meynert) that serve as the primary source of cholinergic input to the entire cortical mantle. Despite its broad involvement in cortical activation, attention, and memory, the functional details of the BF are not well understood due to the anatomical complexity of the region. This study tested the hypothesis that basalocortical connections reflect cortical connectivity patterns. Distinct retrograde tracers were deposited into various frontal and posterior cortical areas, and retrogradely labeled cholinergic and noncholinergic neurons were mapped in the BF. Concurrently, we mapped retrogradely labeled cells in posterior cortical areas that project to various frontal areas, and all cell populations were combined in the same coordinate system. Our studies suggest that the cholinergic and noncholinergic projections to the neocortex are not diffuse, but instead, are organized into segregated or overlapping pools of projection neurons. The extent of overlap between BF populations projecting to the cortex depends on the degree of connectivity between the cortical targets of these projection populations. We suggest that the organization of projections from the BF may enable parallel modulation of multiple groupings of interconnected yet nonadjacent cortical areas.

Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments

Substrates used to culture human embryonic stem cells (hESCs) are typically 2‐dimensional (2‐D) in nature, with limited ability to recapitulate in vivo‐like 3‐dimensional (3‐D) microenvironments. We examined critical determinants of hESC self‐renewal in poly‐d‐lysine‐pretreated synthetic polymer‐based substrates with variable microgeometries, including planar 2‐D films, macroporous 3‐D sponges, and microfibrous 3‐D fiber mats. Completely synthetic 2‐D substrates and 3‐D macroporous scaffolds failed to retain hESCs or support self‐renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self‐renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell‐cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3‐D microgeometries can support hESC colony formation, self‐renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3‐D and for stem cell differentiation in situ and transplantation using defined humanized conditions.—Carlson, A. L., Florek, C. A., Kim, J. J., Neubauer, T., Moore, J. C., Cohen, R. I., Kohn, J., Grumet, M., Moghe, P. V. Microfibrous substrate geometry as a critical trigger for organization, self‐renewal, and differentiation of human embryonic stem cells within synthetic 3‐dimensional microenvironments. FASEB J. 26, 3240–3251 (2012). www.fasebj.org

Vascularization of three-dimensional engineered tissues for regenerative medicine applications.

UNLABELLED Engineering of three-dimensional (3D) tissues is a promising approach for restoring diseased or dysfunctional myocardium with a functional replacement. However, a major bottleneck in this field is the lack of efficient vascularization strategies, because tissue constructs produced in vitro require a constant flow of oxygen and nutrients to maintain viability and functionality. Compared to angiogenic cell therapy and growth factor treatment, bioengineering approaches such as spatial micropatterning, integration of sacrificial materials, tissue decellularization, and 3D bioprinting enable the generation of more precisely controllable neovessel formation. In this review, we summarize the state-of-the-art approaches to develop 3D tissue engineered constructs with vasculature, and demonstrate how some of these techniques have been applied towards regenerative medicine for treatment of heart failure. STATEMENT OF SIGNIFICANCE Tissue engineering is a promising approach to replace or restore dysfunctional tissues/organs, but a major bottleneck in realizing its potential is the challenge of creating scalable 3D tissues. Since most 3D engineered tissues require a constant supply of nutrients, it is necessary to integrate functional vasculature within the tissues in order to facilitate the transport of nutrients. To address these needs, researchers are employing biomaterial engineering and design strategies to foster vessel formation within 3D tissues. This review highlights the state-of-the-art bioengineering tools and technologies to create vascularized 3D tissues for clinical applications in regenerative medicine, highlighting the application of these technologies to engineer vascularized cardiac patches for treatment of heart failure.

Stem cell-based therapies to promote angiogenesis in ischemic cardiovascular disease

Stem cell therapy is a promising approach for the treatment of tissue ischemia associated with myocardial infarction and peripheral arterial disease. Stem and progenitor cells derived from bone marrow or from pluripotent stem cells have shown therapeutic benefit in boosting angiogenesis as well as restoring tissue function. Notably, adult stem and progenitor cells including mononuclear cells, endothelial progenitor cells, and mesenchymal stem cells have progressed into clinical trials and have shown positive benefits. In this review, we overview the major classes of stem and progenitor cells, including pluripotent stem cells, and summarize the state of the art in applying these cell types for treating myocardial infarction and peripheral arterial disease.

11 Mental models of spatial relations and transformations from language

Publisher Summary This chapter focuses on two experiments testing the efficacy of language to evoke spatial transformations. The results support the claim that peoples experience of their bodies interacting with the spatial world underlies their mental representations of space, which in turn support the comprehension of spatial language. The results also add support to the claim that peoples mental representations of space are not like the internalized perceptions of space but depend rather on the peoples conceptions of space. Exploring spatial imagination through description is simpler technically than exploring spatial imagination through experience. In many cases, the effects are the same. Because in the normal course of events people move through stationary environments, it was expected that describing people as stationary and environments as moving would be more difficult to comprehend than describing people as moving in stationary environments. Moreover, research relevant to the nature of mental representations of spatial transformations from discourse come from research on imagery.

Interconnected contribution of tissue morphogenesis and the nuclear protein NuMA to the DNA damage response.

Epithelial tissue morphogenesis is accompanied by the formation of a polarity axis – a feature of tissue architecture that is initiated by the binding of integrins to the basement membrane. Polarity plays a crucial role in tissue homeostasis, preserving differentiation, cell survival and resistance to chemotherapeutic drugs among others. An important aspect in the maintenance of tissue homeostasis is genome integrity. As normal tissues frequently experience DNA double-strand breaks (DSBs), we asked how tissue architecture might participate in the DNA damage response. Using 3D culture models that mimic mammary glandular morphogenesis and tumor formation, we show that DSB repair activity is higher in basally polarized tissues, regardless of the malignant status of cells, and is controlled by hemidesmosomal integrin signaling. In the absence of glandular morphogenesis, in 2D flat monolayer cultures, basal polarity does not affect DNA repair activity but enhances H2AX phosphorylation, an early chromatin response to DNA damage. The nuclear mitotic apparatus protein 1 (NuMA), which controls breast glandular morphogenesis by acting on the organization of chromatin, displays a polarity-dependent pattern and redistributes in the cell nucleus of basally polarized cells upon the induction of DSBs. This is shown using high-content analysis of nuclear morphometric descriptors. Furthermore, silencing NuMA impairs H2AX phosphorylation – thus, tissue polarity and NuMA cooperate to maintain genome integrity.

A high content imaging-based approach for classifying cellular phenotypes.

Current methods to characterize cell-biomaterial interactions are population-based and rely on imaging or biochemical analysis of end-point biological markers. The analysis of stem cells in cultures is further challenged by the heterogeneous nature and divergent fates of stem cells, especially in complex, engineered microenvironments. Here, we describe a high content imaging-based platform capable of identifying cell subpopulations based on cell phenotype-specific morphological descriptors. This method can be utilized to identify microenvironment-responsive morphological descriptors, which can be used to parse cells from a heterogeneous cell population based on emergent phenotypes at the single-cell level and has been successfully deployed to forecast long-term cell lineage fates and screen regenerative phenotype-prescriptive biomaterials.

Optical High Content Nanoscopy of Epigenetic Marks Decodes Phenotypic Divergence in Stem Cells

While distinct stem cell phenotypes follow global changes in chromatin marks, single-cell chromatin technologies are unable to resolve or predict stem cell fates. We propose the first such use of optical high content nanoscopy of histone epigenetic marks (epi-marks) in stem cells to classify emergent cell states. By combining nanoscopy with epi-mark textural image informatics, we developed a novel approach, termed EDICTS (Epi-mark Descriptor Imaging of Cell Transitional States), to discern chromatin organizational changes, demarcate lineage gradations across a range of stem cell types and robustly track lineage restriction kinetics. We demonstrate the utility of EDICTS by predicting the lineage progression of stem cells cultured on biomaterial substrates with graded nanotopographies and mechanical stiffness, thus parsing the role of specific biophysical cues as sensitive epigenetic drivers. We also demonstrate the unique power of EDICTS to resolve cellular states based on epi-marks that cannot be detected via mass spectrometry based methods for quantifying the abundance of histone post-translational modifications. Overall, EDICTS represents a powerful new methodology to predict single cell lineage decisions by integrating high content super-resolution nanoscopy and imaging informatics of the nuclear organization of epi-marks.

Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells

Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV + heparan sulfate + laminin (CHL) or collagen IV + gelatin + heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31+/VE-cadherin+ cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit β3 (>50 fold). To demonstrate the functional importance of integrin β3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin β3-mediated pathway.

Parsing Stem Cell Lineage Development Using High Content Image Analysis of Epigenetic Spatial Markers

This unit describes a protocol for acquiring and analyzing high-content super-resolution images of human stem cell nuclei for the characterization and classification of the cell differentiation paths based on distinct patterns of epigenetic mark organization. Here, we describe the cell culture, immunocytochemical labeling, super-resolution imaging parameters, and MATLAB-based quantitative image analysis approaches for monitoring human mesenchymal stem cells (hMSCs) and human induced pluripotent stem cells (hiPSCs) as the cells differentiate towards various lineages. Although this protocol uses specific cell types as examples, this approach could be easily extended to a variety of cell types and nuclear epigenetic and mechanosensitive biomarkers that are relevant to specific cell developmental scenarios.