Kyle Loh

All Publications

• Lineage-tracing hematopoietic stem cell origins in vivo to efficiently make human HLF+ HOXA+ hematopoietic progenitors from pluripotent stem cells. Developmental cell Fowler, J. L., Zheng, S. L., Nguyen, A., Chen, A., Xiong, X., Chai, T., Chen, J. Y., Karigane, D., Banuelos, A. M., Niizuma, K., Kayamori, K., Nishimura, T., Cromer, M. K., Gonzalez-Perez, D., Mason, C., Liu, D. D., Yilmaz, L., Miquerol, L., Porteus, M. H., Luca, V. C., Majeti, R., Nakauchi, H., Red-Horse, K., Weissman, I. L., Ang, L. T., Loh, K. M. 2024

  Abstract

  The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

  View details for DOI 10.1016/j.devcel.2024.03.003

  View details for PubMedID 38569552

• Monolayer platform to generate and purify primordial germ-like cells in vitro provides insights into human germline specification. Nature communications Vijayakumar, S., Sala, R., Kang, G., Chen, A., Pablo, M. A., Adebayo, A. I., Cipriano, A., Fowler, J. L., Gomes, D. L., Ang, L. T., Loh, K. M., Sebastiano, V. 2023; 14 (1): 5690

  Abstract

  Generating primordial germ cell-like cells (PGCLCs) from human pluripotent stem cells (hPSCs) advances studies of human reproduction and development of infertility treatments, but often entails complex 3D aggregates. Here we develop a simplified, monolayer method to differentiate hPSCs into PGCs within 3.5 days. We use our simplified differentiation platform and single-cell RNA-sequencing to achieve further insights into PGCLC specification. Transient WNT activation for 12h followed by WNT inhibition specified PGCLCs; by contrast, sustained WNT induced primitive streak. Thus, somaticcells (primitive streak) and PGCLCs are related-yet distinct-lineages segregated by temporally-dynamic signaling. Pluripotency factors including NANOG are continuously expressed during the transition from pluripotency to posterior epiblast to PGCs, thus bridging pluripotent and germline states. Finally, hPSC-derived PGCLCs can be easily purified by virtue of their CXCR4+PDGFRA-GARP- surface-marker profile and single-cell RNA-sequencing reveals that they harbor transcriptional similarities with fetal PGCs.

  View details for DOI 10.1038/s41467-023-41302-w

  View details for PubMedID 37709760

• Generating human artery and vein cells from pluripotent stem cells highlights the arterial tropism of Nipah and Hendra viruses. Cell Ang, L. T., Nguyen, A. T., Liu, K. J., Chen, A., Xiong, X., Curtis, M., Martin, R. M., Raftry, B. C., Ng, C. Y., Vogel, U., Lander, A., Lesch, B. J., Fowler, J. L., Holman, A. R., Chai, T., Vijayakumar, S., Suchy, F. P., Nishimura, T., Bhadury, J., Porteus, M. H., Nakauchi, H., Cheung, C., George, S. C., Red-Horse, K., Prescott, J. B., Loh, K. M. 2022

  Abstract

  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. Nature communications Martin, R. M., Fowler, J. L., Cromer, M. K., Lesch, B. J., Ponce, E., Uchida, N., Nishimura, T., Porteus, M. H., Loh, K. M. 2020; 11 (1): 2713

  Abstract

  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 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 PubMedID 27419872

• 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

• Spatially Segregated Macrophage Populations Predict Distinct Outcomes In Colon Cancer. Cancer discovery Matusiak, M., Hickey, J. W., van IJzendoorn, D. G., Lu, G., Kidzinski, L., Zhu, S., Colburg, D. R., Luca, B., Phillips, D. J., Brubaker, S. W., Charville, G. W., Shen, J., Loh, K. M., Okwan-Duodu, D. K., Nolan, G. P., Newman, A. M., West, R. B., van de Rijn, M. 2024

  Abstract

  Tumor-associated macrophages are transcriptionally heterogeneous, but the spatial distribution and cell interactions that shape macrophage tissue roles remain poorly characterized. Here, we spatially resolve five distinct human macrophage populations in normal and malignant human breast and colon tissue and reveal their cellular associations. This spatial map reveals that distinct macrophage populations reside in spatially segregated micro-environmental niches with conserved cellular compositions that are repeated across healthy and diseased tissue. We show that IL4I1+ macrophages phagocytose dying cells in areas with high cell turnover and predict good outcome in colon cancer. In contrast, SPP1+ macrophages are enriched in hypoxic and necrotic tumor regions and portend worse outcome in colon cancer. A subset of FOLR2+ macrophages is embedded in plasma cell niches. NLRP3+ macrophages co-localize with neutrophils and activate an inflammasome in tumors. Our findings indicate that a limited number of unique human macrophage niches function as fundamental building blocks in tissue.

  View details for DOI 10.1158/2159-8290.CD-23-1300

  View details for PubMedID 38552005

• Building human artery and vein endothelial cells from pluripotent stem cells, and enduring mysteries surrounding arteriovenous development. Seminars in cell & developmental biology Loh, K. M., Ang, L. T. 2023

  Abstract

  Owing to their manifold roles in health and disease, there have been intense efforts to synthetically generate blood vessels in vitro from human pluripotent stem cells (hPSCs). However, there are multiple types of blood vessel, including arteries and veins, which are molecularly and functionally different. How can we specifically generate either arterial or venous endothelial cells (ECs) from hPSCs in vitro? Here, we summarize how arterial or venous ECs arise during embryonic development. VEGF and NOTCH arbitrate the bifurcation of arterial vs. venous ECs in vivo. While manipulating these two signaling pathways biases hPSC differentiation towards arterial and venous identities, efficiently generating these two subtypes of ECs has remained challenging until recently. Numerous questions remain to be fully addressed. What is the complete identity, timing and combination of extracellular signals that specify arterial vs. venous identities? How do these extracellular signals intersect with fluid flow to modulate arteriovenous fate? What is a unified definition for endothelial progenitors or angioblasts, and when do arterial vs. venous potentials segregate? How can we regulate hPSC-derived arterial and venous ECs in vitro, and generate organ-specific ECs? In turn, answers to these questions could avail the production of arterial and venous ECs from hPSCs, accelerating vascular research, tissue engineering, and regenerative medicine.

  View details for DOI 10.1016/j.semcdb.2023.06.004

  View details for PubMedID 37393122

• An atlas of lamina-associated chromatin across twelve human cell types reveals an intermediate chromatin subtype. Genome biology Shah, P. P., Keough, K. C., Gjoni, K., Santini, G. T., Abdill, R. J., Wickramasinghe, N. M., Dundes, C. E., Karnay, A., Chen, A., Salomon, R. E., Walsh, P. J., Nguyen, S. C., Whalen, S., Joyce, E. F., Loh, K. M., Dubois, N., Pollard, K. S., Jain, R. 2023; 24 (1): 16

  Abstract

  BACKGROUND: Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamina-associated domains (LADs) are mapped genome-wide in relatively few genetically normal human cell types, which limits our understanding of the role peripheral chromatin plays in development and disease.RESULTS: To address this gap, we map LAMIN B1 occupancy across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas with gene expression and repressive histone modification maps reveal that lamina-associated chromatin in all twelve cell types is organized into at least two subtypes defined by differences in LAMIN B1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Imaging of fluorescently labeled DNA in single cells validates these subtypes and shows radial positioning of LADs with higher LAMIN B1 occupancy and heterochromatic histone modifications primarily embedded within the lamina. In contrast, the second subtype of lamina-associated chromatin is relatively gene dense, accessible, dynamic across development, and positioned adjacent to the lamina. Most genes gain or lose LAMIN B1 occupancy consistent with cell types along developmental trajectories; however, we also identify examples where the enhancer, but not the gene body and promoter, changes LAD state.CONCLUSIONS: Altogether, this atlas represents the largest resource to date for peripheral chromatin organization studies and reveals an intermediate chromatin subtype.

  View details for DOI 10.1186/s13059-023-02849-5

  View details for PubMedID 36691074

• Creating artificial signaling gradients to spatially pattern engineered tissues. Current opinion in biotechnology Zheng, S. L., Loh, K. M. 2022; 78: 102810

  Abstract

  Artificially constructing a fully-fledged tissue - comprising multiple cell types whose identities and spatial arrangements reflect those of a native tissue - remains daunting. There has been impressive progress in generating three-dimensional cell cultures (often dubbed 'organoids') from stem cells. However, it is critical to appreciate that not all such three-dimensional cultures will intrinsically self-organize to spontaneously recreate native tissue architecture. Instead, most tissues in vivo are exogenously patterned by extracellular signaling gradients emanating from organizer cells located outside the tissue. Innovations to impose artificial signaling gradients - using microfluidics, optogenetics, or introducing organizer cells - could thus prove decisive to create spatially patterned tissues in vitro. Additionally, unified terminology to describe these tissue-like simulacra as 'aggregates', 'spheroids', or 'organoids' will be critical for the field.

  View details for DOI 10.1016/j.copbio.2022.102810

  View details for PubMedID 36182872

• A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production. Nature communications Genuth, N. R., Shi, Z., Kunimoto, K., Hung, V., Xu, A. F., Kerr, C. H., Tiu, G. C., Oses-Prieto, J. A., Salomon-Shulman, R. E., Axelrod, J. D., Burlingame, A. L., Loh, K. M., Barna, M. 2022; 13 (1): 5491

  Abstract

  Recent findings suggest that the ribosome itself modulates gene expression. However, whether ribosomes change composition across cell types or control cell fate remains unknown. Here, employing quantitative mass spectrometry during human embryonic stem cell differentiation, we identify dozens of ribosome composition changes underlying cell fate specification. We observe upregulation of RPL10A/uL1-containing ribosomes in the primitive streak followed by progressive decreases during mesoderm differentiation. An Rpl10a loss-of-function allele in mice causes striking early mesodermal phenotypes, including posterior trunk truncations, and inhibits paraxial mesoderm production in culture. Ribosome profiling in Rpl10a loss-of-function mice reveals decreased translation of mesoderm regulators, including Wnt pathway mRNAs, which are also enriched on RPL10A/uL1-containing ribosomes. We further show that RPL10A/uL1 regulates canonical and non-canonical Wnt signaling during stem cell differentiation and in the developing embryo. These findings reveal unexpected ribosome composition modularity that controls differentiation and development through the specialized translation of key signaling networks.

  View details for DOI 10.1038/s41467-022-33263-3

  View details for PubMedID 36123354

• Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling NATURE CHEMICAL BIOLOGY Gonzalez-Perez, D., Das, S., Antfolk, D., Ahsan, H. S., Medina, E., Dundes, C. E., Jokhai, R. T., Egan, E. D., Blacklow, S. C., Loh, K. M., Rodriguez, P. C., Luca, V. C. 2022

  View details for DOI 10.1038/s41589-022-0113-4

  View details for Web of Science ID 000849279700001

• Affinity-matured DLL4 ligands as broad-spectrum modulators of Notch signaling. Nature chemical biology Gonzalez-Perez, D., Das, S., Antfolk, D., Ahsan, H. S., Medina, E., Dundes, C. E., Jokhai, R. T., Egan, E. D., Blacklow, S. C., Loh, K. M., Rodriguez, P. C., Luca, V. C. 2022

  Abstract

  The Notch pathway regulates cell fate decisions and is an emerging target for regenerative and cancer therapies. Recombinant Notch ligands are attractive candidates for modulating Notch signaling; however, their intrinsically low receptor-binding affinity restricts their utility in biomedical applications. To overcome this limitation, we evolved variants of the ligand Delta-like 4 with enhanced affinity and cross-reactivity. A consensus variant with maximized binding affinity, DeltaMAX, binds human and murine Notch receptors with 500- to 1,000-fold increased affinity compared with wild-type human Delta-like 4. DeltaMAX also potently activates Notch in plate-bound, bead-bound and cellular formats. When administered as a soluble decoy, DeltaMAX inhibits Notch in reporter and neuronal differentiation assays, highlighting its dual utility as an agonist or antagonist. Finally, we demonstrate that DeltaMAX stimulates increased proliferation and expression of effector mediators in T cells. Taken together, our data define DeltaMAX as a versatile tool for broad-spectrum activation or inhibition of Notch signaling.

  View details for DOI 10.1038/s41589-022-01113-4

  View details for PubMedID 36050494

• Chimpanzee and pig-tailed macaque iPSCs: Improved culture and generation of primate cross-species embryos. Cell reports Roodgar, M., Suchy, F. P., Nguyen, L. H., Bajpai, V. K., Sinha, R., Vilches-Moure, J. G., Van Bortle, K., Bhadury, J., Metwally, A., Jiang, L., Jian, R., Chiang, R., Oikonomopoulos, A., Wu, J. C., Weissman, I. L., Mankowski, J. L., Holmes, S., Loh, K. M., Nakauchi, H., VandeVoort, C. A., Snyder, M. P. 2022; 40 (9): 111264

  Abstract

  As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.

  View details for DOI 10.1016/j.celrep.2022.111264

  View details for PubMedID 36044843

• Increased ACTL6A occupancy within mSWI/SNF chromatin remodelers drives human squamous cell carcinoma. Molecular cell Chang, C. Y., Shipony, Z., Lin, S. G., Kuo, A., Xiong, X., Loh, K. M., Greenleaf, W. J., Crabtree, G. R. 2021

  Abstract

  Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs.

  View details for DOI 10.1016/j.molcel.2021.10.005

  View details for PubMedID 34687603

• Dach1 Extends Artery Networks and Protects Against Cardiac Injury. Circulation research Raftrey, B., Williams, I. M., Rios Coronado, P. E., Fan, X., Chang, A. H., Zhao, M., Roth, R. K., Trimm, E., Racelis, R., D'Amato, G., Phansalkar, R., Nguyen, A., Chai, T., Gonzalez, K. M., Zhang, Y., Ang, L. T., Loh, K., Bernstein, D., Red-Horse, K. 2021

  Abstract

  Rationale: Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no methods to stimulate artery growth or regeneration in diseased hearts. Studying how arteries are built during development could illuminate strategies for re-building these vessels during ischemic heart disease. We previously found that Dach1 deletion in mouse embryos resulted in small coronary arteries. However, it was not known whether Dach1 gain-of-function would be sufficient to increase arterial vessels and whether this could benefit injury responses. Objective: We investigated how Dach1 overexpression in endothelial cells affected transcription and artery differentiation, and how it influenced recovery from myocardial infarction (MI). Methods and Results: Dach1 was genetically overexpressed in coronary endothelial cells (ECs) in either developing or adult hearts using ApjCreER. This increased the length and number of arterial end branches expanded arteries during development, in both the heart and retina, by inducing capillary ECs to differentiate and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1. In adults, Dach1 overexpression did not cause a statistically significant change artery structure prior to injury, but increased the number of perfused arteries in the injury zone post-MI. Conclusions: Our data demonstrate that increasing Dach1 is a novel method for driving artery specification and extending arterial branches, which could be explored as a means of mitigating the effects of CAD.

  View details for DOI 10.1161/CIRCRESAHA.120.318271

  View details for PubMedID 34383559

• 16p11.2 microdeletion imparts transcriptional alterations in human iPSC-derived models of early neural development. eLife Roth, J. G., Muench, K. L., Asokan, A., Mallett, V. M., Gai, H., Verma, Y., Weber, S., Charlton, C., Fowler, J. L., Loh, K. M., Dolmetsch, R. E., Palmer, T. D. 2020; 9

  Abstract

  Microdeletions and microduplications of the 16p11.2 chromosomal locus are associated with syndromic neurodevelopmental disorders and reciprocal physiological conditions such as macro/microcephaly and high/low body mass index. To facilitate cellular and molecular investigations into these phenotypes, 65 clones of human induced pluripotent stem cells (hiPSCs) were generated from 13 individuals with 16p11.2 copy number variations (CNVs). To ensure these cell lines were suitable for downstream mechanistic investigations, a customizable bioinformatic strategy for the detection of random integration and expression of reprogramming vectors was developed and leveraged towards identifying a subset of 'footprint'-free hiPSC clones. Transcriptomic profiling of cortical neural progenitor cells derived from these hiPSCs identified alterations in gene expression patterns which precede morphological abnormalities reported at later neurodevelopmental stages. Interpreting clinical information-available with the cell lines by request from the Simons Foundation Autism Research Initiative-with this transcriptional data revealed disruptions in gene programs related to both nervous system function and cellular metabolism. As demonstrated by these analyses, this publicly available resource has the potential to serve as a powerful medium for probing the etiology of developmental disorders associated with 16p11.2 CNVs.

  View details for DOI 10.7554/eLife.58178

  View details for PubMedID 33169669

• Bridging naive and primed pluripotency. Nature cell biology Dundes, C. E., Loh, K. M. 2020

  View details for DOI 10.1038/s41556-020-0509-9

  View details for PubMedID 32367045

• Spatially controlled stem cell differentiation via morphogen gradients: A comparison of static and dynamic microfluidic platforms JOURNAL OF VACUUM SCIENCE & TECHNOLOGY A Cui, K. W., Engel, L., Dundes, C. E., Nguyen, T. C., Loh, K. M., Dunn, A. R. 2020; 38 (3)

  View details for DOI 10.1116/1.5142012

  View details for Web of Science ID 000522020800001

• Spatially controlled stem cell differentiation via morphogen gradients: A comparison of static and dynamic microfluidic platforms. Journal of vacuum science & technology. A, Vacuum, surfaces, and films : an official journal of the American Vacuum Society Cui, K. W., Engel, L., Dundes, C. E., Nguyen, T. C., Loh, K. M., Dunn, A. R. 2020; 38 (3): 033205

  Abstract

  The ability to harness the processes by which complex tissues arise during embryonic development would improve the ability to engineer complex tissuelike constructs in vitro-a longstanding goal of tissue engineering and regenerative medicine. In embryos, uniform populations of stem cells are exposed to spatial gradients of diffusible extracellular signaling proteins, known as morphogens. Varying levels of these signaling proteins induce stem cells to differentiate into distinct cell types at different positions along the gradient, thus creating spatially patterned tissues. Here, the authors describe two straightforward and easy-to-adopt microfluidic strategies to expose human pluripotent stem cells in vitro to spatial gradients of desired differentiation-inducing extracellular signals. Both approaches afford a high degree of control over the distribution of extracellular signals, while preserving the viability of the cultured stem cells. The first microfluidic platform is commercially available and entails static culture, whereas the second microfluidic platform requires fabrication and dynamic fluid exchange. In each platform, the authors first computationally modeled the spatial distribution of differentiation-inducing extracellular signals. Then, the authors used each platform to expose human pluripotent stem cells to a gradient of these signals (in this case, inducing a cell type known as the primitive streak), resulting in a regionalized culture with differentiated primitive streak cells predominately localized on one side and undifferentiated stem cells at the other side of the device. By combining this approach with a fluorescent reporter for differentiated cells and live-cell fluorescence imaging, the authors characterized the spatial and temporal dynamics of primitive streak differentiation within the induced signaling gradients. Microfluidic approaches to create precisely controlled morphogen gradients will add to the stem cell and developmental biology toolkit, and may eventually pave the way to create increasingly spatially patterned tissuelike constructs in vitro.

  View details for DOI 10.1116/1.5142012

  View details for PubMedID 32255900

  View details for PubMedCentralID PMC7093209

• Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells. Journal of visualized experiments : JoVE Loh, K. M., Palaria, A., Ang, L. T. 2019

  Abstract

  The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure-which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults-is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

  View details for DOI 10.3791/58975

  View details for PubMedID 31259908

• Antibody Conditioning Enables MHC-Mismatched Hematopoietic Stem Cell Transplants and Organ Graft Tolerance. Cell stem cell George, B. M., Kao, K. S., Kwon, H., Velasco, B. J., Poyser, J., Chen, A., Le, A. C., Chhabra, A., Burnett, C. E., Cajuste, D., Hoover, M., Loh, K. M., Shizuru, J. A., Weissman, I. L. 2019

  Abstract

  Hematopoietic cell transplantation can correct hematological and immunological disorders by replacing a diseased blood system with a healthy one, but this currently requires depleting a patient's existing hematopoietic system with toxic and non-specific chemotherapy, radiation, or both. Here we report an antibody-based conditioning protocol with reduced toxicity and enhanced specificity for robust hematopoietic stem cell (HSC) transplantation and engraftment in recipient mice. Host pre-treatment with six monoclonal antibodies targeting CD47, Tcells, NK cells, and HSCs followed by donor HSC transplantation enabled stable hematopoietic system reconstitution in recipients with mismatches at half (haploidentical) or all major histocompatibility complex (MHC) genes. This approach allowed tolerance to heart tissue from HSC donor strains in haploidentical recipients, showing potential applications for solid organ transplantation without immune suppression. Fully mismatched chimeric mice developed antibody responses to nominal antigens, showing preserved functional immunity. These findings suggest approaches for transplanting immunologically mismatched HSCs and solid organs with limited toxicity.

  View details for DOI 10.1016/j.stem.2019.05.018

  View details for PubMedID 31204177

• Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature Wilkinson, A. C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R. V., Yamamoto, R., Loh, K. M., Nakamura, Y., Watanabe, M., Nakauchi, H., Yamazaki, S. 2019

  Abstract

  Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

  View details for DOI 10.1038/s41586-019-1244-x

  View details for PubMedID 31142833

• A critical look: Challenges in differentiating human pluripotent stem cells into desired cell types and organoids. Wiley interdisciplinary reviews. Developmental biology Fowler, J. L., Ang, L. T., Loh, K. M. 2019: e368

  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.

  View details for DOI 10.1002/wdev.368

  View details for PubMedID 31746148

• 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 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 PubMedID 29466743

• Isolation and 3D expansion of multipotent Sox9(+) mouse lung progenitors NATURE METHODS Nichane, M., Javed, A., Sivakamasundari, V., Ganesan, M., Ang, L., Kraus, P., Lufkin, T., Loh, K. M., Lim, B. 2017; 14 (12): 1205-+

  View details for DOI 10.1038/NMETH.4498

  View details for Web of Science ID 000416604800026

• Isolation and 3D expansion of multipotent Sox9+ mouse lung progenitors. Nature methods Nichane, M., Javed, A., Sivakamasundari, V., Ganesan, M., Ang, L. T., Kraus, P., Lufkin, T., Loh, K. M., Lim, B. 2017; 14 (12): 1205-1212

  Abstract

  Multiple adult tissues are maintained by stem cells of restricted developmental potential which can only form a subset of lineages within the tissue. For instance, the two adult lung epithelial compartments (airways and alveoli) are separately maintained by distinct lineage-restricted stem cells. A challenge has been to obtain multipotent stem cells and/or progenitors that can generate all epithelial cell types of a given tissue. Here we show that mouse Sox9+ multipotent embryonic lung progenitors can be isolated and expanded long term in 3D culture. Cultured Sox9+ progenitors transcriptionally resemble their in vivo counterparts and generate both airway and alveolar cell types in vitro. Sox9+ progenitors that were transplanted into injured adult mouse lungs differentiated into all major airway and alveolar lineages in vivo in a region-appropriate fashion. We propose that a single expandable embryonic lung progenitor population with broader developmental competence may eventually be used as an alternative for region-restricted adult tissue stem cells in regenerative medicine.

  View details for DOI 10.1038/nmeth.4498

  View details for PubMedID 29106405

• Evaluating the regenerative potential and functionality of human liver cells in mice DIFFERENTIATION Tan, A., Loh, K. M., Ang, L. 2017; 98: 25–34

  Abstract

  Liver diseases afflict millions of patients worldwide. Currently, the only long-term treatment for liver failure is the transplantation of a new liver. However, intravenously transplanting a suspension of human hepatocytes might be a less-invasive approach to partially reconstitute lost liver functions in human patients as evinced by promising outcomes in clinical trials. The purpose of this essay is to emphasize outstanding questions that continue to surround hepatocyte transplantation. While adult primary human hepatocytes are the gold standard for transplantation, hepatocytes are heterogeneous. Whether all hepatocytes engraft equally and what specifically defines an "engraftable" hepatocyte capable of long-term liver reconstitution remains unclear. To this end, mouse models of liver injury enable the evaluation of human hepatocytes and their behavior upon transplantation into a complex injured liver environment. While mouse models may not be fully representative of the injured human liver and human hepatocytes tend to engraft mice less efficiently than mouse hepatocytes, valuable lessons have nonetheless been learned from transplanting human hepatocytes into mouse models. With an eye to the future, it will be crucial to eventually detail the optimal biological source (whether in vivo- or in vitro-derived) and presumptive heterogeneity of human hepatocytes and to understand the mechanisms through which they engraft and regenerate liver tissue in vivo.

  View details for PubMedID 29078082

• Live Imaging Reveals that the First Division of Differentiating Human Embryonic Stem Cells Often Yields Asymmetric Fates. Cell reports Brown, K., Loh, K. M., Nusse, R. 2017; 21 (2): 301-307

  Abstract

  How do stem cells respond to signals to initiate differentiation? Here, we show that, despite uniform exposure to differentiation-inducing extracellular signals, individual human embryonic stem cells (hESCs) respond heterogeneously. To track how hESCs incipiently exit pluripotency, we established a system to differentiate hESCs as single cells and conducted live imaging to track their very first cell division. We followed the fate of their earliest daughters as they remained undifferentiated or differentiated toward the primitive streak (the earliest descendants of pluripotent cells). About 30%-50% of the time, hESCs divided to yield one primitive streak and one undifferentiated daughter. The undifferentiated daughter cell was innately resistant to WNT signaling and could not respond to this primitive-streak-specifying differentiation signal. Hence, the first division of differentiating hESCs sometimes yields daughters with diverging fates, with implications for the efficiency of directed differentiation protocols and the underlying rules of lineage commitment.

  View details for DOI 10.1016/j.celrep.2017.09.044

  View details for PubMedID 29020617

• 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. n., Deisseroth, K. n., Luo, L. n. 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 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 PubMedID 27996962

• 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 PubMedID 27869129

• 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

• 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

• 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

• 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

• 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

• 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 PubMedID 23858471

• 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

  View details for PubMedCentralID PMC3701247

• 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