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A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids

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Abstract Too many choices can be problematic. This is certainly the case for human pluripotent stem cells (hPSCs): they harbor the potential to differentiate into hundreds of cell types; yet it is highly challenging to exclusively differentiate hPSCs into a single desired cell type. This review focuses on unresolved and fundamental questions regarding hPSC differentiation and critiquing the identity and purity of the resultant cell populations. These are timely issues in view of the fact that hPSC‐derived cell populations have or are being transplanted into patients in over 30 ongoing clinical trials. While many in vitro differentiation protocols purport to “mimic development,” the exact number and identity of intermediate steps that a pluripotent cell takes to differentiate into a given cell type in vivo remains largely unknown. Consequently, most differentiation efforts inevitably generate a heterogeneous cellular population, as revealed by single‐cell RNA‐sequencing and other analyses. The presence of unwanted cell types in differentiated hPSC populations does not portend well for transplantation therapies. This provides an impetus to precisely control differentiation to desired ends—for instance, by logically blocking the formation of unwanted cell types or by overexpressing lineage‐specifying transcription factors—or by harnessing technologies to selectively purify desired cell types. Conversely, approaches to differentiate three‐dimensional “organoids” from hPSCs intentionally generate heterogeneous cell populations. While this is intended to mimic the rich cellular diversity of developing tissues, whether all such organoids are spatially organized in a manner akin to native organs (and thus, whether they fully qualify as organoids) remains to be fully resolved. This article is categorized under: Adult Stem Cells > Tissue Renewal > Regeneration: Stem Cell Differentiation and Reversion Gene Expression > Transcriptional Hierarchies: Cellular Differentiation Early Embryonic Development: Gastrulation and Neurulation
Unexpected complexities in hPSC differentiation towards the endoderm, mesoderm and ectoderm germ layers. (a) The process by which the definitive endoderm germ layer develops into >12 different organs in vivo is poorly understood (left); areas yet to be fully understood stymie the in vitro differentiation of hPSCs into endodermal derivatives (right). (b) Prevailing strategies to differentiate hPSCs toward pancreatic progenitors are ostensibly divided into several intermediate steps (i), yet outstanding questions remain, including: how the posterior foregut is specified (ii); what the exact identity of the posterior foregut is (i.e., what lineages can it differentiate into) (iii); and whether hPSC‐derived pancreatic progenitors have a dorsal and/or ventral identity (iv). (c) There is no ubiquitous “pan‐mesoderm” progenitor that gives rise to all mesoderm lineages (left), but rather hPSCs differentiate into distinct primitive streak subtypes, each of which gives rise to a distinct mesoderm subtype or alternatively the definitive endoderm (right). (d) The developmental origins of the spinal cord remain unknown, with neural ectoderm and neuromesoderm both serving as potential intermediates for generating spinal cord in vitro (i); recent success in generating substantia nigra dopaminergic neurons was made possible by differentiating hPSCs through a ventral midbrain precursor intermediate instead of a “pan‐neural progenitor” (ii). hPSC, human pluripotent stem cell
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Primitive streak differentiation and the importance of the very first steps of hPSC differentiation. (a) Human pluripotent stem cells (hPSCs) do not directly differentiate into definitive endoderm or mesoderm (i), but first must differentiate through a transitory primitive streak intermediate (ii). (b) In the ~6.5‐day‐old (~E6.5) mouse embryo, there is no “pan‐mesoderm” precursor; rather distinct primitive streak lineages give rise to different types of mesoderm (Lawson, Meneses, & Pedersen, ; Rosenquist, ; Tam & Beddington, ). (c) hPSC‐derived anterior and mid primitive streak populations are broadly marked by both BRACHYURY and MIXL1; however, each primitive streak subtypes has a distinct lineage potential in terms of its ability to further differentiate into downstream cell types
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The merits of heterogeneous cell populations and their relationship to organoids. (a) Differentiating hPSCs into a homogenous monoculture creates purer populations of a given cell type (i), while differentiating cells in a heterogeneous 3D culture provides different cell types the opportunity to reciprocally signal, and mechanically interact, with one another (ii); however whether all 3D cultures meet the strict definition of an “organoid” (Lancaster & Knoblich, ) remains to be determined. (b) Current‐generation hPSC‐derived intestinal organoids rely on the codifferentiation of endoderm and mesoderm derivatives to generate appropriate cellular diversity and spatial organization akin to the native intestine. (c) Current‐generation hPSC‐derived brain organoids possess some key features of early brain development, but various questions remain. hPSC, human pluripotent stem cell
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The virtues of purity and identity during hPSC differentiation. (a) Transplantation of hPSC‐derived heterogeneous populations containing a subset of pancreatic progenitors into rodent models yielded a variety of unwanted cell types including bone and cartilage (Kroon et al., ; Rezania et al., ; Rostovskaya et al., ) (i); at later differentiation stages, assessing the expression of both insulin (an “on‐target” marker) and glucagon (an “off‐target marker) allows for the distinction between nonfunctional polyhormonal cells and β‐cells (ii) (Pagliuca et al., ; Rezania et al., ) (Russ et al., ). (b) Single‐cell RNA‐sequencing of hPSC‐derived endothelial (Paik et al., ) (i), pancreatic (Veres et al., ) (ii), primitive streak (Loh et al., ) (iii), presomitic mesoderm (Loh et al., ) (iii) or lateral mesoderm (Loh et al., ) (iii) populations estimates the purity and the composition of the respective cultures; percentages and cell‐type identities are reported here as indicated in each of the published papers. (c) Enrichment of a particular cell type from a heterogeneous cell population can be accomplished using: (i) FACS, which strictly purifies cell types while adversely affecting cell yield and survival; (ii) magnetic activated cell sorting (MACS), which enriches for cell types with lower purity, but maintains higher cell yield and survival; (iii) cytotoxic antibodies, which generally can deplete one unwanted cell type (e.g., hPSCs), but spare other contaminating cell types; (iv) metabolic selection, which facilitates the selective growth of a desired cell type while also potentially maintaining other contaminating cell types that share the same metabolic growth advantage; (v) and cell‐substrate adhesion which provides favorable conditions for target cells to survive while some, but not all, contaminating cells die. (d) The developmental potential of early hPSC‐derived cell types might be tested by gene expression comparisons with the analogous cell type derived from the human fetus. FACS, fluorescence‐activated cell sorting; hPSC, human pluripotent stem cell
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Expedited differentiation via the overexpression of lineage‐specifying transcription factors. (a) Electrophysiologically active neurons can be generated from hPSCs via directed differentiation using extracellular signals or via overexpression of a neuron‐specifying TF (NGN2) (Zhang et al., ). Do these two differentiation strategies generate equivalent neurons, do the resultant neurons have a clear subtype identity, and how comparable are they to their in vivo counterparts? (b) Overexpression of MYOD1 in hPSCs fails to induce skeletal muscle in the absence of either first differentiating the cells through a mesoderm intermediate (Albini et al., ) or concurrently adding muscle‐inducing extracellular signals (Pawlowski et al., ), suggesting the chromatin landscape decisively dictates the success of TF‐based hPSC differentiation
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Reconstituting cell differentiation and assaying the products thereof. (a) In the first day of hPSC differentiation to endoderm and mesoderm lineages, hPSCs differentiate into anteriormost primitive streak, anterior primitive streak, and mid‐primitive streak, which, respectively, have the competence to further differentiate into definitive endoderm, presomitic mesoderm, or lateral mesoderm, respectively. Subsequent to the primitive streak, manipulation of BMP, TGFβ, and WNT allows guided differentiation into one of these three lineages while suppressing differentiation into unwanted fates (i–iii) (Loh et al., ). (b) Over the course of 4 days of hPSC differentiation into dorsal somites (dermomyotome/future skeletal muscle precursors), WNT specifies four distinct cell types (Chal et al., ; Loh et al., ) and hence dynamic control of WNT signals every 24 hr of differentiation is crucial to progressively differentiate cells along this developmental trajectory
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Early Embryonic Development > Gastrulation and Neurulation
Gene Expression and Transcriptional Hierarchies > Cellular Differentiation
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