Kyle M. Loh

All Publications

• Generating Cellular Diversity and Spatial Form: Wnt Signaling and the Evolution of Multicellular Animals. Developmental cell Loh, K. M., van Amerongen, R., Nusse, R. 2016; 38 (6): 643-655

  Abstract

  There were multiple prerequisites to the evolution of multicellular animal life, including the generation of multiple cell fates ("cellular diversity") and their patterned spatial arrangement ("spatial form"). Wnt proteins operate as primordial symmetry-breaking signals. By virtue of their short-range nature and their capacity to activate both lineage-specifying and cell-polarizing intracellular signaling cascades, Wnts can polarize cells at their site of contact, orienting the axis of cell division while simultaneously programming daughter cells to adopt diverging fates in a spatially stereotyped way. By coupling cell fate to position, symmetry-breaking Wnt signals were pivotal in constructing the metazoan body by generating cellular diversity and spatial form.

  View details for DOI 10.1016/j.devcel.2016.08.011

  View details for PubMedID 27676437

• Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types CELL Loh, K. M., Chen, A., Koh, P. W., Deng, T. Z., Sinha, R., Tsai, J. M., Barkal, A. A., Shen, K. Y., Jain, R., Morganti, R. M., Shyh-Chang, N., Fernhoff, N. B., George, B. M., Wernig, G., Salomon, R. E., Chen, Z., Vogel, H., Epstein, J. A., Kundaje, A., Talbot, W. S., Beachy, P. A., Ang, L. T., Weissman, I. L. 2016; 166 (2): 451-467

  Abstract

  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 Web of Science ID 000380255400021

  View details for PubMedID 27419872

• Ex uno plures: molecular designs for embryonic pluripotency. Physiological reviews Loh, K. M., Lim, B., Ang, L. T. 2015; 95 (1): 245-295

  Abstract

  Pluripotent cells in embryos are situated near the apex of the hierarchy of developmental potential. They are capable of generating all cell types of the mammalian body proper. Therefore, they are the exemplar of stem cells. In vivo, pluripotent cells exist transiently and become expended within a few days of their establishment. Yet, when explanted into artificial culture conditions, they can be indefinitely propagated in vitro as pluripotent stem cell lines. A host of transcription factors and regulatory genes are now known to underpin the pluripotent state. Nonetheless, how pluripotent cells are equipped with their vast multilineage differentiation potential remains elusive. Consensus holds that pluripotency transcription factors prevent differentiation by inhibiting the expression of differentiation genes. However, this does not explain the developmental potential of pluripotent cells. We have presented another emergent perspective, namely, that pluripotency factors function as lineage specifiers that enable pluripotent cells to differentiate into specific lineages, therefore endowing pluripotent cells with their multilineage potential. Here we provide a comprehensive overview of the developmental biology, transcription factors, and extrinsic signaling associated with pluripotent cells, and their accompanying subtypes, in vitro heterogeneity and chromatin states. Although much has been learned since the appreciation of mammalian pluripotency in the 1950s and the derivation of embryonic stem cell lines in 1981, we will specifically emphasize what currently remains unclear. However, the view that pluripotency factors capacitate differentiation, recently corroborated by experimental evidence, might perhaps address the long-standing question of how pluripotent cells are endowed with their multilineage differentiation potential.

  View details for DOI 10.1152/physrev.00001.2014

  View details for PubMedID 25540144

• Stem cell signaling. An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control. Science Clevers, H., Loh, K. M., Nusse, R. 2014; 346 (6205)

  Abstract

  Stem cells fuel tissue development, renewal, and regeneration, and these activities are controlled by the local stem cell microenvironment, the "niche." Wnt signals emanating from the niche can act as self-renewal factors for stem cells in multiple mammalian tissues. Wnt proteins are lipid-modified, which constrains them to act as short-range cellular signals. The locality of Wnt signaling dictates that stem cells exiting the Wnt signaling domain differentiate, spatially delimiting the niche in certain tissues. In some instances, stem cells may act as or generate their own niche, enabling the self-organization of patterned tissues. In this Review, we discuss the various ways by which Wnt operates in stem cell control and, in doing so, identify an integral program for tissue renewal and regeneration.

  View details for DOI 10.1126/science.1248012

  View details for PubMedID 25278615

• Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell stem cell Loh, K. M., Ang, L. T., Zhang, J., Kumar, V., Ang, J., Auyeong, J. Q., Lee, K. L., Choo, S. H., Lim, C. Y., Nichane, M., Tan, J., Noghabi, M. S., Azzola, L., Ng, E. S., Durruthy-Durruthy, J., Sebastiano, V., Poellinger, L., Elefanty, A. G., Stanley, E. G., Chen, Q., Prabhakar, S., Weissman, I. L., Lim, B. 2014; 14 (2): 237-252

  Abstract

  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 Loh, K. M., Lim, B. 2011; 8 (4): 363-369

  Abstract

  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

• Obliterating Obstacles to an Odyssey. Cell stem cell Nichane, M., Loh, K. M. 2018; 23 (3): 313–15

  Abstract

  Why is reprogramming to generate induced pluripotent stem cells (iPSCs) a protracted and inefficient odyssey? In this issue of Cell Stem Cell, Mor etal. (2018) hypothesize that reprogramming factors paradoxically activate and inhibit pluripotency gene expression and show that eliminating Gatad2a (a NuRD corepressor complex subcomponent) rapidly and efficiently reprograms multiple cell types into iPSCs.

  View details for DOI 10.1016/j.stem.2018.08.013

  View details for PubMedID 30193127

• A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells CELL REPORTS Ang, L., Tan, A., Autio, M. I., Goh, S., Choo, S., Lee, K., Tan, J., Pan, B., Lee, J., Lum, J., Lim, C., Yeo, I., Wong, C., Liu, M., Oh, J., Chia, C., Loh, C., Chen, A., Chen, Q., Weissman, I. L., Loh, K. M., Lim, B. 2018; 22 (8): 2190–2205

  Abstract

  How are closely related lineages, including liver, pancreas, and intestines, diversified from a common endodermal origin? Here, we apply principles learned from developmental biology to rapidly reconstitute liver progenitors from human pluripotent stem cells (hPSCs). Mapping the formation of multiple endodermal lineages revealed how alternate endodermal fates (e.g., pancreas and intestines) are restricted during liver commitment. Human liver fate was encoded by combinations of inductive and repressive extracellular signals at different doses. However, these signaling combinations were temporally re-interpreted: cellular competence to respond to retinoid, WNT, TGF-β, and other signals sharply changed within 24 hr. Consequently, temporally dynamic manipulation of extracellular signals was imperative to suppress the production of unwanted cell fates across six consecutive developmental junctures. This efficiently generated 94.1% ± 7.35% TBX3+HNF4A+ human liver bud progenitors and 81.5% ± 3.2% FAH+ hepatocyte-like cells by days 6 and 18 of hPSC differentiation, respectively; the latter improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure.

  View details for DOI 10.1016/j.celrep.2018.01.087

  View details for Web of Science ID 000425489700021

  View details for PubMedID 29466743

  View details for PubMedCentralID PMC5854481

• Thirst-associated preoptic neurons encode an aversive motivational drive. Science (New York, N.Y.) Allen, W. E., DeNardo, L. A., Chen, M. Z., Liu, C. D., Loh, K. M., Fenno, L. E., Ramakrishnan, C., Deisseroth, K., Luo, L. 2017; 357 (6356): 1149–55

  Abstract

  Water deprivation produces a drive to seek and consume water. How neural activity creates this motivation remains poorly understood. We used activity-dependent genetic labeling to characterize neurons activated by water deprivation in the hypothalamic median preoptic nucleus (MnPO). Single-cell transcriptional profiling revealed that dehydration-activated MnPO neurons consist of a single excitatory cell type. After optogenetic activation of these neurons, mice drank water and performed an operant lever-pressing task for water reward with rates that scaled with stimulation frequency. This stimulation was aversive, and instrumentally pausing stimulation could reinforce lever-pressing. Activity of these neurons gradually decreased over the course of an operant session. Thus, the activity of dehydration-activated MnPO neurons establishes a scalable, persistent, and aversive internal state that dynamically controls thirst-motivated behavior.

  View details for DOI 10.1126/science.aan6747

  View details for PubMedID 28912243

• An atlas of transcriptional, chromatin accessibility, and surface marker changes in human mesoderm development SCIENTIFIC DATA Koh, P. W., Sinha, R., Barkal, A. A., Morganti, R. M., Chen, A., Weissman, I. L., Ang, L. T., Kundaje, A., Loh, K. M. 2016; 3

  Abstract

  Mesoderm is the developmental precursor to myriad human tissues including bone, heart, and skeletal muscle. Unravelling the molecular events through which these lineages become diversified from one another is integral to developmental biology and understanding changes in cellular fate. To this end, we developed an in vitro system to differentiate human pluripotent stem cells through primitive streak intermediates into paraxial mesoderm and its derivatives (somites, sclerotome, dermomyotome) and separately, into lateral mesoderm and its derivatives (cardiac mesoderm). Whole-population and single-cell analyses of these purified populations of human mesoderm lineages through RNA-seq, ATAC-seq, and high-throughput surface marker screens illustrated how transcriptional changes co-occur with changes in open chromatin and surface marker landscapes throughout human mesoderm development. This molecular atlas will facilitate study of human mesoderm development (which cannot be interrogated in vivo due to restrictions on human embryo studies) and provides a broad resource for the study of gene regulation in development at the single-cell level, knowledge that might one day be exploited for regenerative medicine.

  View details for DOI 10.1038/sdata.2016.109

  View details for Web of Science ID 000390238600001

  View details for PubMedID 27996962

  View details for PubMedCentralID PMC5170597

• Reprogramming mouse fibroblasts into engraftable myeloerythroid and lymphoid progenitors NATURE COMMUNICATIONS Cheng, H., Ang, H. Y., El Farran, C. A., Li, P., Fang, H. T., Liu, T. M., Kong, S. L., Chin, M. L., Ling, W. Y., Lim, E. K., Li, H., Huber, T., Loh, K. M., Loh, Y., Lim, B. 2016; 7

  Abstract

  Recent efforts have attempted to convert non-blood cells into hematopoietic stem cells (HSCs) with the goal of generating blood lineages de novo. Here we show that hematopoietic transcription factors Scl, Lmo2, Runx1 and Bmi1 can convert a developmentally distant lineage (fibroblasts) into 'induced hematopoietic progenitors' (iHPs). Functionally, iHPs generate acetylcholinesterase(+) megakaryocytes and phagocytic myeloid cells in vitro and can also engraft immunodeficient mice, generating myeloerythoid and B-lymphoid cells for up to 4 months in vivo. Molecularly, iHPs transcriptionally resemble native Kit(+) hematopoietic progenitors. Mechanistically, reprogramming factor Lmo2 implements a hematopoietic programme in fibroblasts by rapidly binding to and upregulating the Hhex and Gfi1 genes within days. Moreover the reprogramming transcription factors also require extracellular BMP and MEK signalling to cooperatively effectuate reprogramming. Thus, the transcription factors that orchestrate embryonic hematopoiesis can artificially reconstitute this programme in developmentally distant fibroblasts, converting them into engraftable blood progenitors.

  View details for DOI 10.1038/ncomms13396

  View details for Web of Science ID 000388078400001

  View details for PubMedID 27869129

  View details for PubMedCentralID PMC5121332

• Inhibition of Apoptosis Overcomes Stage-Related Compatibility Barriers to Chimera Formation in Mouse Embryos. Cell stem cell Masaki, H., Kato-Itoh, M., Takahashi, Y., Umino, A., Sato, H., Ito, K., Yanagida, A., Nishimura, T., Yamaguchi, T., Hirabayashi, M., Era, T., Loh, K. M., Wu, S. M., Weissman, I. L., Nakauchi, H. 2016; 19 (5): 587-592

  Abstract

  Cell types more advanced in development than embryonic stem cells, such as EpiSCs, fail to contribute to chimeras when injected into pre-implantation-stage blastocysts, apparently because the injected cells undergo apoptosis. Here we show that transient promotion of cell survival through expression of the anti-apoptotic gene BCL2 enables EpiSCs and Sox17(+) endoderm progenitors to integrate into blastocysts and contribute to chimeric embryos. Upon injection into blastocyst, BCL2-expressing EpiSCs contributed to all bodily tissues in chimeric animals while Sox17(+) endoderm progenitors specifically contributed in a region-specific fashion to endodermal tissues. In addition, BCL2 expression enabled rat EpiSCs to contribute to mouse embryonic chimeras, thereby forming interspecies chimeras that could survive to adulthood. Our system therefore provides a method to overcome cellular compatibility issues that typically restrict chimera formation. Application of this type of approach could broaden the use of embryonic chimeras, including region-specific chimeras, for basic developmental biology research and regenerative medicine.

  View details for DOI 10.1016/j.stem.2016.10.013

  View details for PubMedID 27814480

• Stem cells: Equilibrium established. Nature Loh, K. M., Lim, B. 2015; 521 (7552): 299-300

  View details for DOI 10.1038/521299a

  View details for PubMedID 25993958

• Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be? REPRODUCTION Roberts, R. M., Loh, K. M., Amita, M., Bernardo, A. S., Adachi, K., Alexenko, A. P., Schust, D. J., Schulz, L. C., Telugu, B. P., Ezashi, T., Pedersen, R. A. 2014; 147 (5): D1-D12

  Abstract

  It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

  View details for DOI 10.1530/REP-14-0080

  View details for Web of Science ID 000336898100001

• Rapid and efficient conversion of integration-free human induced pluripotent stem cells to GMP-grade culture conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

  Abstract

  Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

  View details for DOI 10.1371/journal.pone.0094231

  View details for PubMedID 24718618

  View details for PubMedCentralID PMC3981795

• Rapid and Efficient Conversion of Integration-Free Human Induced Pluripotent Stem Cells to GMP-Grade Culture Conditions. PloS one Durruthy-Durruthy, J., Briggs, S. F., Awe, J., Ramathal, C. Y., Karumbayaram, S., Lee, P. C., Heidmann, J. D., Clark, A., Karakikes, I., Loh, K. M., Wu, J. C., Hoffman, A. R., Byrne, J., Reijo Pera, R. A., Sebastiano, V. 2014; 9 (4)

  View details for DOI 10.1371/journal.pone.0094231

  View details for PubMedID 24718618

• Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chan, C. K., Lindau, P., Jiang, W., Chen, J. Y., Zhang, L. F., Chen, C., Seita, J., Sahoo, D., Kim, J., Lee, A., Park, S., Nag, D., Gong, Y., Kulkarni, S., Luppen, C. A., Theologis, A. A., Wan, D. C., DeBoer, A., Seo, E. Y., Vincent-Tompkins, J. D., Loh, K., Walmsley, G. G., Kraft, D. L., Wu, J. C., Longaker, M. T., Weissman, I. L. 2013; 110 (31): 12643-12648

  Abstract

  Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.

  View details for DOI 10.1073/pnas.1310212110

  View details for Web of Science ID 000322441500042

  View details for PubMedID 23858471

  View details for PubMedCentralID PMC3732968

• Rejuvenating tithonus. EMBO reports Loh, K. M., Lim, B. 2013; 14 (7): 583-584

  View details for DOI 10.1038/embor.2013.81

  View details for PubMedID 23764924

• EPIGENETICS Actors in the cell reprogramming drama NATURE Loh, K. M., Lim, B. 2012; 488 (7413): 599-600

  View details for Web of Science ID 000308095100043

  View details for PubMedID 22932382

• Investigating the bona fide differentiation capacity of human pluripotent stem cells CELL RESEARCH Heng, J. D., Loh, K. M., Ng, H. 2012; 22 (1): 6-8

  View details for DOI 10.1038/cr.2011.142

  View details for Web of Science ID 000299312900003

  View details for PubMedID 21876556

• Recreating Pluripotency? CELL STEM CELL Loh, K. M., Lim, B. 2010; 7 (2): 137-139

  Abstract

  Two Matters Arising articles in this issue challenge the conclusions of a previous Cell Stem Cell paper that found extensive transcriptional differences between hESCs and hiPSCs. The original authors provide a response and set in motion a discussion in the field about appropriate methods for microarray data analysis.

  View details for DOI 10.1016/j.stem.2010.07.005

  View details for Web of Science ID 000281107400002

  View details for PubMedID 20682438

• A Small-Molecule Inhibitor of Tgf-beta Signaling Replaces Sox2 in Reprogramming by Inducing Nanog CELL STEM CELL Ichida, J. K., Blanchard, J., Lam, K., Son, E. Y., Chung, J. E., Egli, D., Loh, K. M., Carter, A. C., Di Giorgio, F. P., Koszka, K., Huangfu, D., Akutsu, H., Liu, D. R., Rubin, L. L., Eggan, K. 2009; 5 (5): 491-503

  Abstract

  The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

  View details for DOI 10.1016/j.stem.2009.09.012

  View details for Web of Science ID 000272019500011

  View details for PubMedID 19818703