A Gutsy Task: Generating Intestinal Tissue from Human Pluripotent Stem Cells

Stacy R. Finkbeiner 0 1 2 Jason R. Spence 0 1 2 0 J. R. Spence Center for Organogenesis, University of Michigan Medical School , Ann Arbor, MI, USA 1 J. R. Spence Department of Cell and Developmental Biology, University of Michigan Medical School , Ann Arbor, MI, USA 2 S. R. Finkbeiner J. R. Spence (&) Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School , Ann Arbor, MI, USA Many significant advances in our understanding of intestine development, intestinal stem cell homeostasis and differentiation have been made in recent years. These advances include novel techniques to culture primary human and mouse intestinal epithelium in threedimensional matrices, and de novo generation of human intestinal tissue from embryonic and induced pluripotent stem cells. This short review will focus on the directed differentiation of human pluripotent stem cells into intestinal tissue, highlight novel uses of this tissue, and compare and contrast this system to primary intestinal epithelial cultures. - Undifferentiated stem cells, which drive embryogenesis and development, are often responsible for adult tissue maintenance and regeneration in response to injury [1]. Stem cells are defined by their long-term ability to divide and self-renew and their ability to give rise to specialized cell types through differentiation. Stem cells are classified by the range of their potential to differentiate into specialized cells. For example, pluripotent stem cells are able to give rise to any embryonic cell type whereas multipotent stem cells can give rise to smaller subsets of more closely related cell types. Adult stem cells are specialized to specific tissues and are often multipotent. They are responsible for tissue homeostasis and regeneration after damage. While they are capable of self-renewal and differentiation, their stemness is reduced over time when grown in vitro [14]. Unlike adult stem cells, pluripotent and embryonic stem cells can give rise to all tissues found in the human body and are virtually unlimited in their ability to proliferate in vitro, making them attractive for use in biomedical research and regenerative medicine. Human pluripotent stem cells (hPSCs) broadly refer to all pluripotent human stem cell types, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). hESCs are most efficiently derived from the inner cell mass of human blastocytes generated during in vitro fertilization procedures [5]. They are commonly grown in culture on either inactivated feeder layers of murine embryonic fibroblasts or under feeder-free conditions on tissue culture plates coated with a basement membrane protein substrate [57]. The development of conditions for feeder-free derivation and growth of hESCs [6, 811] has enabled standardized production of cells more amenable for use in therapeutic applications (reviewed elsewhere [12]). One major challenge when using hESC-derived cells for therapeutic purposes is generating tissue for transplantation that will not be rejected by the patients immune system. Another challenge for the use of hESCs has been the ethical opposition to destroying human embryos during hESC isolation and accordingly, strict rules exist regarding derivation and use of new hESC lines in projects reliant on US federal funding. Recently, methods to reprogram human or mouse adult-like somatic cells into embryonic-like stem cells was described by Shinya Yamanaka, a seminal discovery awarded the Nobel Prize in Medicine in 2012 [13, 14]. This method, called cellular reprogramming, was used to generate mouse and human iPSCs from fibroblast cells through the forced expression of a specific set of transcription factors (Oct4, Sox2, Klf4, c-Myc) [1518]. These reprogrammed cells resemble hESCs in morphology and stem cell-defining characteristics, although detailed comparative gene expression studies suggest that there may be important differences between hESCs and iPSCs (reviewed elsewhere [19]). The original protocol for iPSC generation involved use of retroviral vectors which leave a genetic footprint in the reprogrammed cell, raising concerns for the safety of using iPSCs for transplantation [16]. In recent years, the methods have been further refined to eliminate the use of retroviruses [18], thus making it possible to generate patient-specific iPSCs without changing the genetic composition of the cells. hPSCs, embryonic and induced, can now be used to study and recapitulate many embryonic developmental stages in vitro. Translational embryology is an emerging field in which researchers are able to use what we know about different stages of embryonic development to direct differentiation of pluripotent stem cells into specialized cells and tissues (reviewed elsewhere [20, 21]). Directed differentiation is achieved by treating cells with recombinant proteins or small molecules that regulate important developmental signaling pathways, thereby mimicking key events during in vivo development. This approach has been used to generate a wide spectrum of cell types derived from all three primary germ layers (ectoderm, mesoderm, and endoderm), holding great promise for studies and treatments of many diseases [21, 22]. This review focuses on recent advances that used directed differentiation to generate intestinal tissue from hPSCs. Growing the Intestine In Vivo Differentiation of hESCs or iPSCs into intestinal tissue requires a step-wise process mimicking major developmental events including definitive endoderm differentiation, gut specification and morphogenesis, and intestine development, growth, and homeostasis. We will briefly highlight these major developmental milestones. The three primary germ layers, generated in the process of gastrulation during embryogenesis, all contribute to the development of the intestine. The enteric nervous system that innervates the gut arises from the ectoderm; the smooth muscle, connective tissue, and vasculature of the gut arise from the mesoderm; and intestinal epithelium arises from the endoderm [2326]. For the purpose of this review, we will focus on the development of the intestinal epithelium. The TGF-b superfamily member Nodal is essential for the specification of endoderm. Variable levels of exposure to this growth factor control anteriorposterior (AP) patterning of the endoderm [27]. Fibroblast growth factor (FGF), Wnt, bone morphogenetic protein (BMP), and retinoic acid signaling are also involved in AP patterning and induction of tissue-specific transcription factors (reviewed elsewhere [23, 28]). Simultaneous with formation of the gut tube, the endoderm is specified into future intestinal epithelium primarily through the induction of the transcription factor Cdx2 [2932]. At this stage, the intestine is a single layer of Cdx2? cuboidal epithelial cells that transitions to become a pseudostratified epithelium. The subsequent rapid expansion and thickening (...truncated)