Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Merkle, F. T., A. Maroof, T. Wataya, Y. Sasai, L. Studer, K. Eggan, and A. F. Schier. 2015. “Generation of Neuropeptidergic Hypothalamic Neurons from Human Pluripotent Stem Cells.” Development 142 (4): 633–43. https://doi.org/10.1242/dev.117978. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41461212 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 633-643 doi:10.1242/dev.117978 RESEARCH ARTICLE STEM CELLS AND REGENERATION Generation of neuropeptidergic hypothalamic neurons from human pluripotent stem cells Florian T. Merkle1,2,3,4, Asif Maroof1,3,4, Takafumi Wataya5,6, Yoshiki Sasai5, Lorenz Studer7,8, Kevin Eggan1,2,3,4,* and Alexander F. Schier2,3,* ABSTRACT neurons has been linked to common diseases, such as obesity, Hypothalamic neurons orchestrate many essential physiological hypertension, mood disorders and sleep disorders, that place a and behavioral processes via secreted neuropeptides, and are substantial burden on Western healthcare systems (Meyer- relevant to human diseases such as obesity, narcolepsy and Lindenberg et al., 2011; Spiegelman and Flier, 2001; Swaab, infertility. We report the differentiation of human pluripotent stem 1999, 2006; Wataya et al., 2008) (supplementary material Table S1). cells into many of the major types of neuropeptidergic hypothalamic To enable the study and therapeutic use of human hypothalamic neurons, including those producing pro-opiolemelanocortin, agouti- neurons, we aimed to generate these cells in vitro from human ‘ related peptide, hypocretin/orexin, melanin-concentrating hormone, pluripotent stem cells (hPSCs) using two distinct approaches: self- ’ oxytocin, arginine vasopressin, corticotropin-releasing hormone patterning and directed differentiation. The self-patterning (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can approach permits organ-like tissue development in vitro via the be generated using a ‘self-patterning’ strategy that yields a broad array cell-cell and paracrine interactions that pattern tissues in vivo of cell types, or via a more reproducible directed differentiation (Ludwig and Thomson, 2007; Sasai et al., 2012). Self-patterning is approach. Stem cell-derived human hypothalamic neurons share a rational choice for hypothalamic differentiation as pluripotent characteristic morphological properties and gene expression patterns stem cells are predisposed to generate anterior neural structures such with their counterparts in vivo, and are able to integrate into the mouse as the hypothalamus (Puelles and Rubenstein, 2003; Watanabe brain. These neurons could form the basis of cellular models, chemical et al., 2007) by default (Kamiya et al., 2011; Wilson and screens or cellular therapies to study and treat common human Rubenstein, 2000) (Fig. 1A). Directed differentiation of hPSCs in β diseases. the presence of inhibitors of the TGF /NODAL/activin and BMP signaling pathways leads to the efficient production of neural KEY WORDS: Differentiation, Hypothalamus, Narcolepsy, progenitors (Blinkov and Glezer, 1968; Chambers et al., 2009) that Neuropeptide, Pluripotent, Stem cell, Mouse, Human can be patterned into ventral forebrain neurons by the early inhibition of the WNT signaling pathway followed by activation of INTRODUCTION the sonic hedgehog (SHH) pathway (Maroof et al., 2013; Meyer- Although it makes up only 0.3% of the adult human brain, the Lindenberg et al., 2011; Spiegelman and Flier, 2001; Swaab, 1999, hypothalamus is one of its most complex and essential regions 2006). We reasoned that a similar approach could be taken to (Blinkov and Glezer, 1968; Maroof et al., 2013). In their central generate human hypothalamic neurons (Fig. 1D). location in the ventral forebrain (Fig. 1A,B), hypothalamic neurons Here, we report the differentiation of both human embryonic stem are positioned to sense both neural and physiological signals and to cells (hESCs) and human induced pluripotent stem cells (hiPSCs) respond by releasing neurotransmitters and peptide neuromodulators into hypothalamic neurons using complementary self-patterning into the brain. Furthermore, they can induce the release of hormones and directed differentiation approaches. The neuropeptide- from the endocrine glands to regulate physiology and behavior. expressing cells we observed are highly enriched or exclusively These activities are relegated to different subtypes of hypothalamic localized in the hypothalamus and were morphologically similar to neurons that secrete distinct neuropeptides to regulate puberty, their in vivo counterparts. The efficiency with which these rare reproduction, stress, circadian rhythms and immune function, as well neuropeptidergic cell types were produced in vitro rivaled their as more complex behaviors, including sleep, mood and social prevalence in the human hypothalamus in vivo. Furthermore, human behavior (Fig. 1C; supplementary material Table S1). As these hypothalamic neurons survived transplantation into the mouse brain functions are fundamental, the dysfunction of human hypothalamic and continued to express hypothalamic neuropeptides in ectopic locations. The resources described here lay the foundation for the 1 study of human hypothalamic neurons in health and disease. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. 2Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. 3Harvard Stem Cell Institute, RESULTS Harvard University, Cambridge, MA 02138, USA. 4Stanley Center for Psychiatric Research, Broad Institute of Harvard and MIT, Cambridge, MA 02138, USA. Self-patterning of hPSCs into hypothalamic progenitors 5Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan. 6Department of To generate hypothalamic progenitors from hPSCs, we took 7 Neurosurgery, Hospital for Sick Children, Toronto ON M5G 1X8, Canada. The advantage of the natural propensity of pluripotent stem cells to Center for Stem Cell Biology, Sloan-Kettering Institute for Cancer Research, New York, NY 10065, USA. 8Developmental Biology Program, Sloan-Kettering differentiate into anterior neural structures, such as the Institute for Cancer Research, New York, NY 10065, USA. hypothalamus (Fig. 1A-C), in the absence of inductive signals (Fig. 1D). We first implemented a previously described self- *Authors for correspondence ([email protected]; [email protected]) patterning protocol (Sasai et al., 2012; Wataya et al., 2008) to confirm the generation of hypothalamic progenitors from mouse Received 23 September 2014; Accepted 12 December 2014 embryonic stem cells (mESCs) plated in a growth factor-free, DEVELOPMENT 633 RESEARCH ARTICLE Development (2015) 142, 633-643 doi:10.1242/dev.117978 Fig. 1. In vivo location and in vitro generation of hypothalamic neurons. (A,B) Schematic lateral views of the embryonic mammalian brain (A) and adult human brain (B) showing the anterior and ventral location of the hypothalamus (red). (C) Enlarged schematic of the human hypothalamus showing the approximate distribution of select neuropeptidergic cell types (colored dots). MCH, melanin- concentrating hormone; AVP, arginine vasopressin; TRH, thyrotropin-releasing hormone; HCRT, hypocretin (also known as orexin); POMC, pro-opiomelanocortin; CRH, corticotropin-releasing hormone; OXT, oxytocin; AGRP, agouti-related protein. (D) Self-patterning and directed differentiation strategies for hypothalamic differentiation from hPSCs. gfCDM, growth factor-free chemically defined minimal medium; LDN, 100 nM LDN-193189; SB, 10 μM SB435142; XAV, 2 μM XAV939; SAG, 1 μM smoothened agonist; Pur, 1 μM purmorphamine; BMP, bone morphogenetic protein; TGFβ, transforming growth factor β; WNT, wingless-related MMTV integration site; SHH, sonic hedgehog. chemically defined medium (gfCDM). We observed that mESCs separated from the ventricle-like structures by at least 50 μm seeded into gfCDM differentiated into neurons that expressed (Fig. 2D,E), as is seen in the embryonic nervous system (Marín and hypothalamus-specific neuropeptides, including hypocretin Rubenstein, 2003). Together, our findings indicated that hPSCs (supplementary material Fig. S1). could self-pattern into aggregates that resembled the embryonic To adapt the mESC self-patterning protocol to hPSCs, we grew neuroepithelium. two hESC and two hiPSC lines (Fig. 2A) under feeder-free To determine whether cells within self-patterned cell aggregates conditions in the defined medium mTeSR (Ludwig and Thomson, adopted a hypothalamic identity, we cryosectioned cell aggregates at 2007; Puelles and Rubenstein, 2003). When hPSCs were D30 and performed immunostaining for the transcription factors dissociated and plated into gfCDM, they died within several days, forkhead box G1 (FOXG1), which is expressed throughout the even in the presence of the Rho kinase (ROCK) inhibitor Y27632 telencephalon but is absent from the hypothalamus (Tao and Lai, (Kamiya et al., 2011; Watanabe et al., 2007; Wilson and Rubenstein, 1992), and NK2 homeobox 1 (NKX2.1), which is expressed