Kyle Loh, PhD
Our goal is to understand how various human tissues are constructed from stem cells during development: we apply reductionist approaches to developmental biology. We are delineating a roadmap for early human tissue development by defining the branching lineage choices through which human embryonic and induced pluripotent stem cells develop into >20 different tissue progenitors. At each branching lineage choice where stem cells choose to differentiate into one of two lineages, we are discovering the extracellular cues that induce one cell-type or another. By applying a combination of the relevant inductive signals while blocking the repressive signals that otherwise induce the alternate fate, we can "force" stem cells to differentiate into a relatively pure population of a given cell-type - thus fulfilling a key goal of regenerative medicine. This has allowed us to generate enriched populations of human liver, bone and heart progenitors from pluripotent stem cells, each of which could regenerate their cognate human tissue in respective mouse models. Our current objectives are two-fold: (1) to reconstitute the development of relatively pure populations of human tissue progenitors in vitro for regenerative medicine and (2) to understand the molecular basis of the developmental competence of stem cells and how they subsequently assemble into tissues.
Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses.
Stem cell research endeavors to generate specific subtypes of classically defined "cell types." Here, we generate >90% pure human artery or vein endothelial cells from pluripotent stem cells within 3-4 days. We specified artery cells by inhibiting vein-specifying signals and vice versa. These cells modeled viral infection of human vasculature by Nipah and Hendra viruses, which are extraordinarily deadly (∼57%-59% fatality rate) and require biosafety-level-4 containment. Generating pure populations of artery and vein cells highlighted that Nipah and Hendra viruses preferentially infected arteries; arteries expressed higher levels of their viral-entry receptor. Virally infected artery cells fused into syncytia containing up to 23 nuclei, which rapidly died. Despite infecting arteries and occupying ∼6%-17% of their transcriptome, Nipah and Hendra largely eluded innate immune detection, minimally eliciting interferon signaling. We thus efficiently generate artery and vein cells, introduce stem-cell-based toolkits for biosafety-level-4 virology, and explore the arterial tropism and cellular effects of Nipah and Hendra viruses.
View details for DOI 10.1016/j.cell.2022.05.024
View details for PubMedID 35738284
Improving the safety of human pluripotent stem cell therapies using genome-edited orthogonal safeguards.
2020; 11 (1): 2713
Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.
View details for DOI 10.1038/s41467-020-16455-7
View details for PubMedID 32483127
Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types
2016; 166 (2): 451-467
Stem-cell differentiation to desired lineages requires navigating alternating developmental paths that often lead to unwanted cell types. Hence, comprehensive developmental roadmaps are crucial to channel stem-cell differentiation toward desired fates. To this end, here, we map bifurcating lineage choices leading from pluripotency to 12 human mesodermal lineages, including bone, muscle, and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation toward unwanted fates and rapidly steer pluripotent stem cells toward 80%-99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously unobservable human embryonic event transiently marked by HOPX expression. Collectively, this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes. VIDEO ABSTRACT.
View details for DOI 10.1016/j.cell.2016.06.011
View details for PubMedID 27419872
Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations.
Cell stem cell
2014; 14 (2): 237-252
Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of "pre-enhancer" states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation.
View details for DOI 10.1016/j.stem.2013.12.007
View details for PubMedID 24412311
A Precarious Balance: Pluripotency Factors as Lineage Specifiers
CELL STEM CELL
2011; 8 (4): 363-369
Understanding the basis of the unrestricted multilineage differentiation potential of pluripotent cells will be of developmental and translational consequence. We propose that pluripotency transcription factors are lineage specifiers that direct commitment to specific fetal lineages. Individual factors bestow the ability to differentiate into particular cell types, and concomitant expression of multiple lineage specifiers within pluripotent cells enables differentiation into every fetal lineage. Moreover, we speculate that, rather than being an intrinsically stable "ground state," pluripotency is an inherently precarious condition in which rival lineage specifiers continually compete to specify differentiation along mutually exclusive lineages.
View details for DOI 10.1016/j.stem.2011.03.013
View details for Web of Science ID 000289707100008
View details for PubMedID 21474100