Scientific Resources

Obtaining highly purified differentiated cells via directed differentiation from human pluripotent stem cells (hPSCs) is an essential step for their clinical application. Among the various conditions that should be optimized,the precise role and contribution of the extracellular matrix (ECM) during differentiation are relatively unclear. Here,using a short fragment of laminin 411 (LM411-E8),an ECM predominantly expressed in the vascular endothelial basement membrane,we demonstrate that the directed switching of defined ECMs robustly yields highly-purified (textgreater95%) endothelial progenitor cells (PSC-EPCs) without cell sorting from hPSCs in an integrin-laminin axis-dependent manner. Single-cell RNA-seq analysis revealed that LM411-E8 resolved intercellular transcriptional heterogeneity and escorted the progenitor cells to the appropriate differentiation pathway. The PSC-EPCs gave rise to functional endothelial cells both in vivo and in vitro. We therefore propose that sequential switching of defined matrices is an important concept for guiding cells towards desired fate. View Publication

During adaptive immune responses,CD8(+) T cells with low TCR affinities are released early into the circulation before high-affinity clones become dominant at later time points. How functional avidity maturation is orchestrated in lymphoid tissue and how low-affinity cells contribute to host protection remains unclear. In this study,we used intravital imaging of reactive lymph nodes (LNs) to show that T cells rapidly attached to dendritic cells irrespective of TCR affinity,whereas one day later,the duration of these stable interactions ceased progressively with lowering peptide major histocompatibility complex (pMHC) affinity. This correlated inversely BATF (basic leucine zipper transcription factor,ATF-like) and IRF4 (interferon-regulated factor 4) induction and timing of effector differentiation,as low affinity-primed T cells acquired cytotoxic activity earlier than high affinity-primed ones. After activation,low-affinity effector CD8(+) T cells accumulated at efferent lymphatic vessels for egress,whereas high affinity-stimulated CD8(+) T cells moved to interfollicular regions in a CXCR3-dependent manner for sustained pMHC stimulation and prolonged expansion. The early release of low-affinity effector T cells led to rapid target cell elimination outside reactive LNs. Our data provide a model for affinity-dependent spatiotemporal orchestration of CD8(+) T cell activation inside LNs leading to functional avidity maturation and uncover a role for low-affinity effector T cells during early microbial containment. View Publication

Mucosa-associated invariant T (MAIT) cells are a unique innate T cell subset that is necessary for rapid recruitment of activated CD4(+) T cells to the lungs after pulmonary F. tularensis LVS infection. Here,we investigated the mechanisms behind this effect. We provide evidence to show that MAIT cells promote early differentiation of CCR2-dependent monocytes into monocyte-derived DCs (Mo-DCs) in the lungs after F. tularensis LVS pulmonary infection. Adoptive transfer of Mo-DCs to MAIT cell-deficient mice (MR1(-/-) mice) rescued their defect in the recruitment of activated CD4(+) T cells to the lungs. We further demonstrate that MAIT cell-dependent GM-CSF production stimulated monocyte differentiation in vitro,and that in vivo production of GM-CSF was delayed in the lungs of MR1(-/-) mice. Finally,GM-CSF-deficient mice exhibited a defect in monocyte differentiation into Mo-DCs that was phenotypically similar to MR1(-/-) mice. Overall,our data demonstrate that MAIT cells promote early pulmonary GM-CSF production,which drives the differentiation of inflammatory monocytes into Mo-DCs. Further,this delayed differentiation of Mo-DCs in MR1(-/-) mice was responsible for the delayed recruitment of activated CD4(+) T cells to the lungs. These findings establish a novel mechanism by which MAIT cells function to promote both innate and adaptive immune responses. View Publication

A fundamental impediment to understanding the brain is the availability of inexpensive and robust methods for targeting and manipulating specific neuronal populations. The need to overcome this barrier is pressing because there are considerable anatomical,physiological,cognitive and behavioral differences between mice and higher mammalian species in which it is difficult to specifically target and manipulate genetically defined functional cell types. In particular,it is unclear the degree to which insights from mouse models can shed light on the neural mechanisms that mediate cognitive functions in higher species,including humans. Here we describe a novel recombinant adeno-associated virus that restricts gene expression to GABAergic interneurons within the telencephalon. We demonstrate that the viral expression is specific and robust,allowing for morphological visualization,activity monitoring and functional manipulation of interneurons in both mice and non-genetically tractable species,thus opening the possibility to study GABAergic function in virtually any vertebrate species. View Publication

The AP-1 factor basic leucine zipper transcription factor,ATF-like (BATF) is important for CD4(+) Th17,Th9,and follicular Th cell development. However,its precise role in Th2 differentiation and function remains unclear,and the requirement for BATF in nonallergic settings of type-2 immunity has not been explored. In this article,we show that,in response to parasitic helminths,Batf(-/-) mice are unable to generate follicular Th and Th2 cells. As a consequence,they fail to establish productive type-2 immunity during primary and secondary infection. Batf(-/-) CD4(+) T cells do not achieve type-2 cytokine competency,which implies that BATF plays a key role in the regulation of IL-4 and IL-13. In contrast to Th17 and Th9 cell subsets in which BATF binds directly to promoter and enhancer regions to regulate cytokine expression,our results show that BATF is significantly enriched at Rad50 hypersensitivity site (RHS)6 and RHS7 of the locus control region relative to AP-1 sites surrounding type-2 cytokine loci in Th2 cells. Indeed,Batf(-/-) CD4(+) T cells do not obtain permissive epigenetic modifications within the Th2 locus,which were linked to RHS6 and RHS7 function. In sum,these findings reveal BATF as a central modulator of peripheral and humoral hallmarks of type-2 immunity and begin to elucidate a novel mechanism by which it regulates type-2 cytokine production through its modification of the Th2 locus control region. View Publication

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G textgreater A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study,here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts,the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G textgreater A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast,adding BMP4 at later times decreased iPSC generation. ID genes,transcriptional targets of BMP-SMAD signaling,were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence,a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes. View Publication

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections,but the inhibitors and activators involved have not been fully delineated. Here,we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis,cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further,we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells,but not Fas expression by B cells. Thus,cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency. View Publication

Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However,treatment is restricted to corneal transplantation,which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study,hiPSCs were successfully differentiated into neural crest cells (NCCs),the embryonic precursor to keratocytes,and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies. View Publication

Peripheral blood was collected from a 39-year-old unaffected female carrier of an X-linked recessive mutation of Iduronate 2-sulfatase gene (NM000202.7(IDS):c.85CtextgreaterT) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC showed normal karyotype. The line offers a good platform to study MPS II pathophysiology,for drug testing,early biomarker discovery and gene therapy studies. View Publication

Peripheral blood was collected from a 1-year-old male patient with an X-linked recessive mutation of Iduronate 2-sulfatase (IDS) gene (NM000202.7(IDS):c.85CtextgreaterT) causing MPS II (OMIM 309900). Peripheral blood mononuclear cells (PBMCs) were reprogrammed by lentiviral delivery of a self-silencing hOKSM polycistronic vector. The pluripotency of the iPSC line was confirmed by the expression of pluripotency-associated markers and in vitro spontaneous differentiation towards the 3 germ layers. The iPSC line showed normal karyotype. The cell line offers a good platform to study MPS II pathophysiology,for drug testing,early biomarker discovery and gene therapy studies. View Publication

A new method of spatially controlled gene regulation in 3D-cultured human embryonic stem cells is developed using hollow gold nanoshells (HGNs) and near-infrared (NIR) light. Targeted cell(s) are discriminated from neighboring cell(s) by focusing NIR light emitted from a two-photon microscope. Irradiation of cells that have internalized HGNs releases surface attached siRNAs and leads to concomitant gene downregulation. View Publication

New genetic tools are needed to understand the functional interactions between HIV and human host factors in primary cells. We recently developed a method to edit the genome of primary CD4(+) T cells by electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs). Here,we adapted this methodology to a high-throughput platform for the efficient,arrayed editing of candidate host factors. CXCR4 or CCR5 knockout cells generated with this method are resistant to HIV infection in a tropism-dependent manner,whereas knockout of LEDGF or TNPO3 results in a tropism-independent reduction in infection. CRISPR/Cas9 RNPs can furthermore edit multiple genes simultaneously,enabling studies of interactions among multiple host and viral factors. Finally,in an arrayed screen of 45 genes associated with HIV integrase,we identified several candidate dependency/restriction factors,demonstrating the power of this approach as a discovery platform. This technology should accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. View Publication