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

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

Translating Embryogenesis to Generate Organoids: Novel Approaches to Personalized Medicine

“…Spontaneous differentiation due to long-term culture can generate unwanted lineages ( Tang et al., 2019 ) and the high frequency of contaminating progenitors compared with the desired cell types during in vitro differentiation of PSCs can affect organoid reproducibility ( Veres et al., 2019 ; Yao et al., 2017 ). Therefore “separating the wheat from the chaff” is important to generate organoids, as a minor population of undifferentiated human PSCs has a higher chance to give rise to teratoma (tumor) that often out-competes organ reconstitution in vivo ( Fowler et al., 2020 ). Heterogeneity of both differentiated and undifferentiated PSCs is a common problem, and therefore using specific cell surface markers can efficiently generate the desired lineage.…”

Organotypic Modeling of the Tumor Landscape

“…However, the lack of standardization and quality control of stem cell culture are an obstacle for their use in clinical studies. The use of pluripotent stem cells for organoid generation can be hampered by the presence of contaminating progenitors that can yield undesired cell types and a small population of undifferentiated PSCs can give rise to tumors that outcompete organ reconstitution in vivo (Fowler et al, 2020). Furthermore, due to different culture methods, organoids may present undesired phenotypic variabilities.…”

Modeling of Hypoxic Brain Injury through 3D Human Neural Organoids

“…Organoids derived from human pluripotent stem cells are widely expected to fill the remaining gaps between animal models and humans, as stem cells derived from humans are the main sources of cultured organoid [20,21]. The development of research platforms using organoid models takes less time than the establishment of animal models.…”