Richard I. Sherwood

Isolation of Adult Mouse Myogenic Progenitors:Functional Heterogeneity of Cells within and Engrafting Skeletal Muscle

Skeletal muscle regeneration in adults is thought to occur through the action of myogenic satellite cells located in close association with mature muscle fibers; however, these precursor cells have not been prospectively isolated, and recent studies have suggested that additional muscle progenitors, including cells of bone marrow or hematopoietic origin, may exist. To clarify the origin(s) of adult myogenic cells, we used phenotypic, morphological, and functional criteria to identify and prospectively isolate a subset of myofiber-associated cells capable at the single cell level of generating myogenic colonies at high frequency. Importantly, although muscle-engrafted cells from marrow and/or circulation localized to the same anatomic compartment as myogenic satellite cells and expressed some though not all satellite cell markers, they displayed no intrinsic myogenicity. Together, these studies describe the clonal isolation of functional adult myogenic progenitors and demonstrate that these cells do not arise from hematopoietic or other bone marrow or circulating precursors.

Sox17 promotes differentiation in mouse embryonic stem cells by directly regulating extraembryonic gene expression and indirectly antagonizing self-renewal

In embryonic stem (ES) cells, a well-characterized transcriptional network promotes pluripotency and represses gene expression required for differentiation. In comparison, the transcriptional networks that promote differentiation of ES cells and the blastocyst inner cell mass are poorly understood. Here, we show that Sox17 is a transcriptional regulator of differentiation in these pluripotent cells. ES cells deficient in Sox17 fail to differentiate into extraembryonic cell types and maintain expression of pluripotency-associated transcription factors, including Oct4, Nanog, and Sox2. In contrast, forced expression of Sox17 down-regulates ES cell-associated gene expression and directly activates genes functioning in differentiation toward an extraembryonic endoderm cell fate. We show these effects of Sox17 on ES cell gene expression are mediated at least in part through a competition between Sox17 and Nanog for common DNA-binding sites. By elaborating the function of Sox17, our results provide insight into how the transcriptional network promoting ES cell self-renewal is interrupted, allowing cellular differentiation.

Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.

Transcriptional dynamics of endodermal organ formation

Although endodermal organs including the liver, pancreas, and intestine are of significant therapeutic interest, the mechanism by which the endoderm is divided into organ domains during embryogenesis is not well understood. To better understand this process, global gene expression profiling was performed on early endodermal organ domains. This global analysis was followed up by dynamic immunofluorescence analysis of key transcription factors, uncovering novel expression patterns as well as cell surface proteins that allow prospective isolation of specific endodermal organ domains. Additionally, a repressive interaction between Cdx2 and Sox2 was found to occur at the prospective stomach–intestine border, with the hepatic and pancreatic domains forming at this boundary, and Hlxb9 was revealed to have graded expression along the dorsal–ventral axis. These results contribute to understanding the mechanism of endodermal organogenesis and should assist efforts to replicate this process using pluripotent stem cells. Developmental Dynamics 238:29–42, 2009.

Determinants of Skeletal Muscle Contributions from Circulating Cells, Bone Marrow Cells, and Hematopoietic Stem Cells

To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c‐kit+Thy1.1loLin−Sca‐1+ hemato‐poietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker–expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor‐derived myofibers expressed both donor‐specific and host‐specific markers, suggesting that they arise by low‐level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage.

High-throughput mapping of regulatory DNA

Quantifying the effects of cis-regulatory DNA on gene expression is a major challenge. Here, we present the multiplexed editing regulatory assay (MERA), a high-throughput CRISPR-Cas9–based approach that analyzes the functional impact of the regulatory genome in its native context. MERA tiles thousands of mutations across ∼40 kb of cis-regulatory genomic space and uses knock-in green fluorescent protein (GFP) reporters to read out gene activity. Using this approach, we obtain quantitative information on the contribution of cis-regulatory regions to gene expression. We identify proximal and distal regulatory elements necessary for expression of four embryonic stem cell–specific genes. We show a consistent contribution of neighboring gene promoters to gene expression and identify unmarked regulatory elements (UREs) that control gene expression but do not have typical enhancer epigenetic or chromatin features. We compare thousands of functional and nonfunctional genotypes at a genomic location and identify the base pair–resolution functional motifs of regulatory elements.

Wnt signaling specifies and patterns intestinal endoderm

Wnt signaling has been implicated in many developmental processes, but its role in early endoderm development is not well understood. Wnt signaling is active in posterior endoderm as early as E7.5. Genetic and chemical activation show that the Wnt pathway acts directly on endoderm to induce the intestinal master regulator Cdx2, shifting global gene away from anterior endoderm and toward a posterior, intestinal program. In a mouse embryonic stem cell differentiation platform that yields pure populations of definitive endoderm, Wnt signaling induces intestinal gene expression in all cells. We have identified a set of genes specific to the anterior small intestine, posterior small intestine, and large intestine during early development, and show that Wnt, through Cdx2, activates large intestinal gene expression at high doses and small intestinal gene expression at lower doses. These findings shed light on the mechanism of embryonic intestinal induction and provide a method to manipulate intestinal development from embryonic stem cells.

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states

The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.

Genetic targeting of the endoderm with claudin-6CreER

A full description of the ontogeny of the β cell would guide efforts to generate β cells from embryonic stem cells (ESCs). The first step requires an understanding of definitive endoderm: the genes and signals responsible for its specification, proliferation, and patterning. This report describes a global marker of definitive endoderm, Claudin‐6 (Cldn6). We report its expression in early development with particular attention to definitive endoderm derivatives. To create a genetic system to drive gene expression throughout the definitive endoderm with both spatial and temporal control, we target the endogenous locus with an inducible Cre recombinase (Cre‐ERT2) cassette. Cldn6 null mice are viable and fertile with no obvious phenotypic abnormalities. We also report a lineage analysis of the fate of Cldn6‐expressing embryonic cells, which is relevant to the development of the pancreas, lung, and liver. Developmental Dynamics 237:504–512, 2008.

Regulation and function of skeletal muscle stem cells.

Skeletal muscle satellite cells, which reside beneath the basal lamina of mature muscle fibers, function as myogenic precursors and are required for normal muscle growth and repair. Satellite cells share a common anatomical localization, yet they exhibit substantial phenotypic and functional heterogeneity. Recent efforts in the field of adult myogenesis have been aimed at dissecting this heterogeneity and reveal the presence of discrete cell lineages within the muscle that function independently and interactively to maintain muscle homeostasis and to determine the outcome of muscle damage. Normal developmental regulation of the frequency and function of these distinct tissue precursors, and pathological deregulation of their activity, may have an important role in age- and disease-dependent loss of muscle regenerative activity.