David A. Brafman

Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces

Realization of the full potential of human pluripotent stem cells (hPSCs) in regenerative medicine requires the development of well-defined culture conditions for their long-term growth and directed differentiation. Current practices for maintaining hPSCs generally utilize empirically determined combinations of feeder cells and other animal-based products, which are expensive, difficult to isolate, subject to batch-to-batch variations, and unsuitable for cell-based therapies. Using a high-throughput screening approach, we identified several polymers that can support self-renewal of hPSCs. While most of these polymers provide support for only a short period of time, we identified a synthetic polymer poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-alt-MA) that supported the long-term attachment, proliferation and self-renewal of HUES1, HUES9, and iPSCs. The hPSCs cultured on PMVE-alt-MA maintained their characteristic morphology, expressed high levels of markers of pluripotency, and retained a normal karyotype. Such cost-effective, polymer-based matrices that support long-term self-renewal and proliferation of hPSCs will not only help to accelerate the translational perspectives of hPSCs, but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation.

Defining Long-Term Maintenance Conditions of Human Embryonic Stem Cells With Arrayed Cellular Microenvironment Technology

The optimization of defined growth conditions is necessary for the development of clinical application of human embryonic stem cells (hESCs). Current research has focused on developing defined media formulations for long-term culture of hESCs with little attention on the establishment of defined substrates for hESC proliferation and self-renewal. Presently available technologies are insufficient to address the full complement of factors that may regulate hESC proliferation and maintenance of pluripotency. Here, we report the application of a multifactorial array technology to identify fully defined and optimized culture conditions for the proliferation of hESCs. Through the systematic screening of extracellular matrix proteins (ECMPs) and other signaling molecules, we developed and characterized a completely defined culture system for the long-term self-renewal of three independent hESC lines. In the future, the novel array platform and analysis procedure presented here will be applied toward the directed differentiation of hESCs and maintenance of other stem and progenitor cell populations.

Regulation of endodermal differentiation of human embryonic stem cells through integrin-ECM interactions

Many cellular responses during development are regulated by interactions between integrin receptors and extracellular matrix proteins (ECMPs). Although the majority of recent studies in human embryonic stem cell (hESC) differentiation have focused on the role of growth factors, such as FGF, TGFβ, and WNT, relatively little is known about the role of ECMP-integrin signaling in this process. Moreover, current strategies to direct hESC differentiation into various lineages are inefficient and have yet to produce functionally mature cells in vitro. This suggests that additional factors, such as ECMPs, are required for the efficient differentiation of hESCs. Using a high-throughput multifactorial cellular array technology, we investigated the effect of hundreds of ECMP combinations and concentrations on differentiation of several hPSC lines to definitive endoderm (DE), an early embryonic cell population fated to give rise to internal organs such as the lung, liver, pancreas, stomach, and intestine. From this screen we identified fibronectin (FN) and vitronectin (VTN) as ECMP components that promoted DE differentiation. Analysis of integrin expression revealed that differentiation toward DE led to an increase in FN-binding integrin α5 (ITGA5) and VTN-binding integrin αV (ITGAV). Conditional short hairpin RNA-mediated knockdown of ITGA5 and ITGAV disrupted hESC differentiation toward DE. Finally, fluorescence-based cell sorting for ITGA5 and ITGAV significantly enriched cells with gene expression signatures associated with DE, demonstrating that these cell surface proteins permit isolation and enrichment of DE from hESCs. These data provide evidence that FN and VTN promote endoderm differentiation of hESCs through interaction with ITGA5 and ITGAV, and that ECMP-integrin interactions are required for hESC differentiation into functionally mature cells.

The WNT receptor FZD7 is required for maintenance of the pluripotent state in human embryonic stem cells

Significance Embryonic stem cells (ESCs) are unique in their ability to expand and self-renew indefinitely while retaining the potential to give rise to all mature cell types. The molecular mechanisms underlying these properties remain poorly understood. We investigated the role of the highly conserved WNT signaling pathway in controlling self-renewal and found that the WNT receptor encoded by the frizzled family receptor 7 (FZD7) gene is essential for maintaining human ESCs in an undifferentiated and pluripotent state. Using an FZD7-specific fragment antigen binding protein, as well as knockdown of FZD7 expression, we showed that the FZD7 receptor transduces a WNT/β-catenin signal in human ESCs. These data demonstrate that an endogenous WNT signaling loop is essential for the maintenance of human ESCs in an undifferentiated state. WNT signaling is involved in maintaining stem cells in an undifferentiated state; however, it is often unclear which WNTs and WNT receptors are mediating these activities. Here we examined the role of the WNT receptor FZD7 in maintaining human embryonic stem cells (hESCs) in an undifferentiated and pluripotent state. FZD7 expression is significantly elevated in undifferentiated cells relative to differentiated cell populations, and interfering with its expression or function, either by short hairpin RNA-mediated knockdown or with a fragment antigen binding (Fab) molecule directed against FZD7, disrupts the pluripotent state of hESCs. The FZD7-specific Fab blocks signaling by Wnt3a protein by down-regulating FZD7 protein levels, suggesting that FZD7 transduces Wnt signals to activate Wnt/β-catenin signaling. These results demonstrate that FZD7 encodes a regulator of the pluripotent state and that hESCs require endogenous WNT/β-catenin signaling through FZD7 to maintain an undifferentiated phenotype.

Arrayed cellular microenvironments for identifying culture and differentiation conditions for stem, primary and rare cell populations.

During the development of an organism, cells are exposed to a myriad of signals, structural components and scaffolds, which collectively make up the cellular microenvironment. The majority of current developmental biology studies examine the effect of individual or small subsets of molecules and parameters on cellular behavior, and they consequently fail to explore the complexity of factors to which cells are exposed. Here we describe a technology, referred to as arrayed cellular microenvironments (ACMEs), that allows for a high-throughput examination of the effects of multiple extracellular components in a combinatorial manner on any cell type of interest. We will specifically focus on the application of this technology to human pluripotent stem cells (hPSCs), a population of cells with tremendous therapeutic potential, and one for which growth and differentiation conditions are poorly characterized and far from defined and optimized. A standard ACME screen uses the technologies previously applied to the manufacture and analysis of DNA microarrays, requires standard cell-culture facilities and can be performed from beginning to end within 5–10 days.

PNIPAAm-based biohybrid injectable hydrogel for cardiac tissue engineering

UNLABELLED Injectable biomaterials offer a non-invasive approach to deliver cells into the myocardial infarct region to maintain a high level of cell retention and viability and initiate the regeneration process. However, previously developed injectable matrices often suffer from low bioactivity or poor mechanical properties. To address this need, we introduced a biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with excellent bioactivity as well as mechanical robustness for cardiac tissue engineering. A unique feature of our work was that we performed extensive in vitro biological analyses to assess the functionalities of cardiomyocytes (CMs) alone and in co-culture with cardiac fibroblasts (CFs) (2:1 ratio) within the hydrogel matrix. The synthesized hydrogel exhibited viscoelastic behavior (storage modulus: 1260 Pa) and necessary water content (75%) to properly accommodate the cardiac cells. The encapsulated cells demonstrated a high level of cell survival (90% for co-culture condition, day 7) and spreading throughout the hydrogel matrix in both culture conditions. A dense network of stained F-actin fibers (∼ 6 × 10(4) μm(2) area coverage, co-culture condition) illustrated the formation of an intact and three dimensional (3D) cell-embedded matrix. Furthermore, immunostaining and gene expression analyses revealed mature phenotypic characteristics of cardiac cells. Notably, the co-culture group exhibited superior structural organization and cell-cell coupling, as well as beating behavior (average ∼ 45 beats per min, co-culture condition, day 7). The outcome of this study is envisioned to open a new avenue for extensive in vitro characterization of injectable matrices embedded with 3D mono- and co-culture of cardiac cells prior to in vivo experiments. STATEMENT OF SIGNIFICANCE In this work, we synthesized a new class of biohybrid temperature-responsive poly(N-isopropylacrylamide) PNIPAAm-Gelatin-based injectable hydrogel with suitable bioactivity and mechanical properties for cardiac tissue engineering. A significant aspect of our work was that we performed extensive in vitro biological analyses to assess the functionality of cardiomyocytes alone and in co-culture with cardiac fibroblasts encapsulated within the 3D hydrogel matrix.

Endogenous WNT Signaling Regulates hPSC-Derived Neural Progenitor Cell Heterogeneity and Specifies Their Regional Identity

Summary Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population that is capable of nearly indefinite expansion and subsequent differentiation into the various neuronal and supporting cell types that comprise the CNS. However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS. In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation. Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures. The ability to manipulate WNT signaling to generate regionally specific NPCs and neurons will be useful for studying human neural development and will greatly enhance the translational potential of hPSCs for neural-related therapies.

Constructing stem cell microenvironments using bioengineering approaches

Within the adult organism, stem cells reside in defined anatomical microenvironments called niches. These architecturally diverse microenvironments serve to balance stem cell self-renewal and differentiation. Proper regulation of this balance is instrumental to tissue repair and homeostasis, and any imbalance can potentially lead to diseases such as cancer. Within each of these microenvironments, a myriad of chemical and physical stimuli interact in a complex (synergistic or antagonistic) manner to tightly regulate stem cell fate. The in vitro replication of these in vivo microenvironments will be necessary for the application of stem cells for disease modeling, drug discovery, and regenerative medicine purposes. However, traditional reductionist approaches have only led to the generation of cell culture methods that poorly recapitulate the in vivo microenvironment. To that end, novel engineering and systems biology approaches have allowed for the investigation of the biological and mechanical stimuli that govern stem cell fate. In this review, the application of these technologies for the dissection of stem cell microenvironments will be analyzed. Moreover, the use of these engineering approaches to construct in vitro stem cell microenvironments that precisely control stem cell fate and function will be reviewed. Finally, the emerging trend of using high-throughput, combinatorial methods for the stepwise engineering of stem cell microenvironments will be explored.

Biomaterial Approaches for Stem Cell-Based Myocardial Tissue Engineering

Adult and pluripotent stem cells represent a ready supply of cellular raw materials that can be used to generate the functionally mature cells needed to replace damaged or diseased heart tissue. However, the use of stem cells for cardiac regenerative therapies is limited by the low efficiency by which stem cells are differentiated in vitro to cardiac lineages as well as the inability to effectively deliver stem cells and their derivatives to regions of damaged myocardium. In this review, we discuss the various biomaterial-based approaches that are being implemented to direct stem cell fate both in vitro and in vivo. First, we discuss the stem cell types available for cardiac repair and the engineering of naturally and synthetically derived biomaterials to direct their in vitro differentiation to the cell types that comprise heart tissue. Next, we describe biomaterial-based approaches that are being implemented to enhance the in vivo integration and differentiation of stem cells delivered to areas of cardiac damage. Finally, we present emerging trends of using stem cell-based biomaterial approaches to deliver pro-survival factors and fully vascularized tissue to the damaged and diseased cardiac tissue.

The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

In order to efficiently edit eukaryotic genomes, it is critical to test the impact of chromatin dynamics on CRISPR/Cas9 function and develop strategies to adapt the system to eukaryotic contexts. So far, research has extensively characterized the relationship between the CRISPR endonuclease Cas9 and the composition of the RNA-DNA duplex that mediates the systems precision. Evidence suggests that chromatin modifications and DNA packaging can block eukaryotic genome editing by custom-built DNA endonucleases like Cas9; however, the underlying mechanism of Cas9 inhibition is unclear. Here, we demonstrate that closed, gene-silencing-associated chromatin is a mechanism for the interference of Cas9-mediated DNA editing. Our assays use a transgenic cell line with a drug-inducible switch to control chromatin states (open and closed) at a single genomic locus. We show that closed chromatin inhibits binding and editing at specific target sites and that artificial reversal of the silenced state restores editing efficiency. These results provide new insights to improve Cas9-mediated editing in human and other mammalian cells.