Cell-specific alterations in Pitx1 regulatory landscape activation caused by the loss of a single enhancer.

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.

CTLA-4Ig Improves Hyperalgesia in a Mouse Model of Osteoporosis.

This study aimed to evaluate skeletal pain associated with osteoporosis and to examine the inhibitory effects of cytotoxic T lymphocyte-associated antigen-4Ig (CTLA-4Ig) administration in ovariectomized (OVX) mice. Eight-week-old female ddY mice were assigned to three groups: sham-operated mice (SHAM) treated with vehicle, OVX mice treated with vehicle (OVX), and OVX mice treated with CTLA-4Ig (CTLA-4Ig). Vehicle or CTLA-4Ig was injected intraperitoneally, starting immediately after surgery. After 4 weeks of treatment, mechanical sensitivity was examined, and the bilateral hind limbs were removed and evaluated by micro-computed tomography, immunohistochemical analyses, and messenger RNA expression analysis. Ovariectomy induced bone loss and mechanical hyperalgesia in the hindlimbs. CTLA-4Ig treatment prevented bone loss in the hindlimbs compared to vehicle administration in the OVX group. Moreover, mechanical hyperalgesia was significantly decreased in the CTLA-4Ig treatment group in comparison to the OVX group. The expression levels of tumor necrosis factor-α (TNF-α) and sclerostin (SOST), as well as the number of osteoclasts, were increased, and the expression level of Wnt-10b was decreased in the OVX group compared with the SHAM group, whereas these parameters were improved in the CTLA-4Ig group compared with the OVX group. The novelty of this research is that CTLA-4Ig administration prevented bone loss and mechanical hyperalgesia induced by ovariectomy in the hindlimbs.

Geminin is required for Hox gene regulation to pattern the developing limb.

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.

Knockout of beta-2 microglobulin reduces stem cell-induced immune rejection and enhances ischaemic hindlimb repair via exosome/miR-24/Bim pathway.

Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M- UMSCs in which human leucocyte antigen (HLA) light chain ß2-microglobulin (B2M) was deleted. The therapeutic potential of B2M- UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M- UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR-24 as a major component of the exosomes originating from B2M- UMSCs. We identified Bim as a potential target of miR-24 through bioinformatics analysis, which was further confirmed by loss-of-function and gain-of-function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs-induced immune rejection, and it provides a universal clinical-scale cell source for tissue repair and regeneration without the need for HLA matching in the future.

Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure.

Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.

miR-210 Enhances the Therapeutic Potential of Bone-Marrow-Derived Circulating Proangiogenic Cells in the Setting of Limb Ischemia.

Therapies based on circulating proangiogenic cells (PACs) have shown promise in ischemic disease models but require further optimization to reach the bedside. Ischemia-associated hypoxia robustly increases microRNA-210 (miR-210) expression in several cell types, including endothelial cells (ECs). In ECs, miR-210 represses EphrinA3 (EFNA3), inducing proangiogenic responses. This study provides new mechanistic evidences for a role of miR-210 in PACs. PACs were obtained from either adult peripheral blood or cord blood. miR-210 expression was modulated with either an inhibitory complementary oligonucleotide (anti-miR-210) or a miRNA mimic (pre-miR-210). Scramble and absence of transfection served as controls. As expected, hypoxia increased miR-210 in PACs. In vivo, migration toward and adhesion to the ischemic endothelium facilitate the proangiogenic actions of transplanted PACs. In vitro, PAC migration toward SDF-1α/CXCL12 was impaired by anti-miR-210 and enhanced by pre-miR-210. Moreover, pre-miR-210 increased PAC adhesion to ECs and supported angiogenic responses in co-cultured ECs. These responses were not associated with changes in extracellular miR-210 and were abrogated by lentivirus-mediated EFNA3 overexpression. Finally, ex-vivo pre-miR-210 transfection predisposed PACs to induce post-ischemic therapeutic neovascularization and blood flow recovery in an immunodeficient mouse limb ischemia model. In conclusion, miR-210 modulates PAC functions and improves their therapeutic potential in limb ischemia.

Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice.

Catch-up growth after insults to growing organs is paramount to achieving robust body proportions. In fly larvae, injury to individual tissues is followed by local and systemic compensatory mechanisms that allow the damaged tissue to regain normal proportions with other tissues. In vertebrates, local catch-up growth has been described after transient reduction of bone growth, but the underlying cellular responses are controversial. We developed an approach to study catch-up growth in foetal mice in which mosaic expression of the cell cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right limb serves as an internal control. Unexpectedly, left-right limb symmetry remained normal, revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an orchestrated response was triggered involving local enhancement of bone growth and systemic growth reduction that ensured that body proportions were maintained. The local response entailed hyperproliferation of spared left limb chondrocytes that was associated with reduced chondrocyte density. The systemic effect involved impaired placental function and IGF signalling, revealing bone-placenta communication. Therefore, vertebrates, like invertebrates, can mount coordinated local and systemic responses to developmental insults that ensure that normal body proportions are maintained.

Robust Revascularization in Models of Limb Ischemia Using a Clinically Translatable Human Stem Cell-Derived Endothelial Cell Product.

Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31+/CD144+), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy.

Leg-local neural mechanisms for searching and learning enhance robotic locomotion.

Adapting motor output based on environmental forces is critical for successful locomotion in the real world. Arthropods use at least two neural mechanisms to adjust muscle activation while walking based on detected forces. Mechanism 1 uses negative feedback of leg depressor force to ensure that each stance leg supports an appropriate amount of the body's weight. Mechanism 2 encourages searching for ground contact if the leg supports no body weight. We expand the neural controller for MantisBot, a robot based upon a praying mantis, to include these mechanisms by incorporating leg-local memory and command neurons, as observed in arthropods. We present results from MantisBot transitioning between searching and stepping, mimicking data from animals as reported in the literature.

Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules.

Isl1 is required for two processes during hindlimb development: initiation of the processes directing hindlimb development in the lateral plate mesoderm and configuring posterior hindlimb field in the nascent hindlimb buds. During these processes, Isl1 expression is restricted to the posterior mesenchyme of hindlimb buds. How this dynamic change in Isl1 expression is regulated remains unknown. We found that two evolutionarily conserved sequences, located 3' to the Isl1 gene, regulate LacZ transgene expression in the hindlimb-forming region in mouse embryos. Both sequences contain GATA binding motifs, and expression pattern analysis identified that Gata6 is expressed in the flank and the anterior portion of nascent hindlimb buds. Recent studies have shown that conditional inactivation of Gata6 in mice causes hindlimb-specific pre-axial polydactyly, indicating a role of Gata6 in anterior-posterior patterning of hindlimbs. We studied whether Gata6 restricts Isl1 in the nascent hindlimb bud through the cis-regulatory modules. In vitro experiments demonstrate that GATA6 binds to the conserved GATA motifs in the cis-regulatory modules. GATA6 repressed expression of a luciferase reporter that contains the cis-regulatory modules by synergizing with Zfpm2. Analyses of Gata6 mutant embryos showed that ISL1 levels are higher in the anterior of nascent hindlimb buds than in wild type. Moreover, we detected a greater number of Isl1-transcribing cells in the anterior of nascent hindlimb buds in Gata6 mutants. Our results support a model in which Gata6 contributes to repression of Isl1 expression in the anterior of nascent hindlimb buds.

PITX1 promotes chondrogenesis and myogenesis in mouse hindlimbs through conserved regulatory targets.

The PITX1 transcription factor is expressed during hindlimb development, where it plays a critical role in directing hindlimb growth and the specification of hindlimb morphology. While it is known that PITX1 regulates hindlimb formation, in part, through activation of the Tbx4 gene, other transcriptional targets remain to be elucidated. We have used a combination of ChIP-seq and RNA-seq to investigate enhancer regions and target genes that are directly regulated by PITX1 in embryonic mouse hindlimbs. In addition, we have analyzed PITX1 binding sites in hindlimbs of Anolis lizards to identify ancient PITX1 regulatory targets. We find that PITX1-bound regions in both mouse and Anolis hindlimbs are strongly associated with genes implicated in limb and skeletal system development. Gene expression analyses reveal a large number of misexpressed genes in the hindlimbs of Pitx1-/- mouse embryos. By intersecting misexpressed genes with genes that have neighboring mouse PITX1 binding sites, we identified 440 candidate targets of PITX1. Of these candidates, 68 exhibit ultra-conserved PITX1 binding events that are shared between mouse and Anolis hindlimbs. Among the ancient targets of PITX1 are important regulators of cartilage and skeletal muscle development, including Sox9 and Six1. Our data suggest that PITX1 promotes chondrogenesis and myogenesis in the hindlimb by direct regulation of several key members of the cartilage and muscle transcriptional networks.

Pitx1 determines characteristic hindlimb morphologies in cartilage micromass culture.

The shapes of homologous skeletal elements in the vertebrate forelimb and hindlimb are distinct, with each element exquisitely adapted to their divergent functions. Many of the signals and signalling pathways responsible for patterning the developing limb bud are common to both forelimb and hindlimb. How disparate morphologies are generated from common signalling inputs during limb development remains poorly understood. We show that, similar to what has been shown in the chick, characteristic differences in mouse forelimb and hindlimb cartilage morphology are maintained when chondrogenesis proceeds in vitro away from the endogenous limb bud environment. Chondrogenic nodules that form in high-density micromass cultures derived from forelimb and hindlimb buds are consistently different in size and shape. We described analytical tools we have developed to quantify these differences in nodule morphology and demonstrate that characteristic hindlimb nodule morphology is lost in the absence of the hindlimb-restricted limb modifier gene Pitx1. Furthermore, we show that ectopic expression of Pitx1 in the forelimb is sufficient to generate nodule patterns characteristic of the hindlimb. We also demonstrate that hindlimb cells are less adhesive to the tissue culture substrate and, within the limb environment, to the extracellular matrix and to each other. These results reveal autonomously programmed differences in forelimb and hindlimb cartilage precursors of the limb skeleton are controlled, at least in part, by Pitx1 and suggest this has an important role in generating distinct limb-type morphologies. Our results demonstrate that the micromass culture system is ideally suited to study cues governing morphogenesis of limb skeletal elements in a simple and experimentally tractable in vitro system that reflects in vivo potential.

Level-specific amputations and resulting regenerative outcomes in the mouse distal phalanx.

Mouse digit tip regeneration involves an intricate coordinated regrowth of the terminal phalanx, nail, dermis and epidermis. During this time, regenerating digits undergo wound healing, blastema formation, and differentiation. However, the regenerative response of the digit is dependent on the level of the amputation. Amputation of <30% of the distal phalanx (P3), with part of the base nail remaining, results in extensive digit regeneration. In contrast, >60% P3 removal results in no regeneration. This level-dependent regenerative ability of the mouse digit provides a comparative model between regeneration and non-regeneration that may enable identification of specific factors critical to regeneration. Although the ability to create regenerating and non-regenerating conditions has been well established, the regenerative response between these regions ("intermediate" zone) has received less scrutiny, and may add insight to the regenerative processes, including the degree of histolysis, and the level of blastema formation. The objective of this study is then to compare the regeneration capacity between amputation levels within the regenerating (<30%), intermediate (40-59%), and non-regenerating (>60%) regions. Results indicated that regenerative and intermediate amputations led to significant histolysis and blastema formation of the distal phalanx 14 days post-amputation. Unlike the regenerating digits, intermediate amputations led to incomplete regeneration whereby regrowth of the digits were not to the levels of the intact or regenerating digits. Non-regenerating amputations did not exhibit significant histolysis or blastema formation. Remarkably, the histolytic process resulted in day 14 P3 lengths that were similar regardless of the initial amputation over 19%. The differences in histolysis, blastema formation and injury outcomes were also marked by changes in the number of proliferating cells and osteoclasts. Altogether, these results indicate that although intermediate amputations result in histolysis and blastema formation similar to regenerating digits, the resulting cellular composition of the blastema differs, contributing to incomplete regeneration.

Production of endothelial progenitor cells from skin fibroblasts by direct reprogramming for clinical usages.

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14 d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12 ± 0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

Cellular and molecular drivers of differential organ growth: insights from the limbs of Monodelphis domestica.

A fundamental question in biology is "how is growth differentially regulated during development to produce organs of particular sizes?" We used a new model system for the study of differential organ growth, the limbs of the opossum (Monodelphis domestica), to investigate the cellular and molecular basis of differential organ growth in mammals. Opossum forelimbs grow much faster than hindlimbs, making opossum limbs an exceptional system with which to study differential growth. We first used the great differences in opossum forelimb and hindlimb growth to identify cellular processes and molecular signals that underlie differential limb growth. We then used organ culture and pharmacological addition of FGF ligands and inhibitors to test the role of the Fgf/Mitogen-activated protein kinases (MAPK) signaling pathway in driving these cellular processes. We found that molecular signals from within the limb drive differences in cell proliferation that contribute to the differential growth of the forelimb and hindlimbs of opossums. We also found that alterations in the Fgf/MAPK pathway can generate differences in cell proliferation that mirror those observed between wild-type forelimb and hindlimbs of opossums and that manipulation of Fgf/MAPK signaling affects downstream focal adhesion-extracellular matrix (FA-ECM) and Wnt signaling in opossum limbs. Taken together, these findings suggest that evolutionary changes in the Fgf/MAPK pathway could help drive the observed differences in cell behaviors and growth in opossum forelimb and hindlimbs.

Postnatal training of 129/Sv mice confirms the long-term influence of early exercising on the motor properties of mice.

A previous study showed that motor experiences during critical periods of development durably affect the motor properties of adult C57BL/6J mice. However, dependence on early environmental features may vary with the genetic profile. To evaluate the contribution of the genetic background on external influences to motricity, we performed the same experiment in a 129/Sv mouse strain that show a strongly different motor profile. Mice were subjected to endurance training (enriched environment or forced treadmill), hypergravity (chronic centrifugation), or simulated microgravity (hindlimb unloading) between postnatal days 10 and 30. They were then returned to standard housing until testing at the age of nine months. The endurance-trained mice showed a fast-slow shift in the deep zone of the tibialis. In addition, mice reared in the enriched environment showed a modified gait and body posture, and improved performance on the rotarod, whereas forced treadmill training did not affect motor output. Hypergravity induced a fast-slow shift in the superficial zone of the tibialis, with no consequence on motor output. Hindlimb unloading provoked an increased percentage of immature hybrid fibres in the tibialis and a shift in the soleus muscle. When compared with similarly reared C57BL/6J mice, 129/Sv mice showed qualitative differences attributable to the lower efficiency of early training due to their lower basal motor activity level. Nevertheless, the results are essentially consistent in both strains, and support the hypothesis that early motor experience influences the muscle phenotype and motor output.

Comparison of fetal cartilage-derived progenitor cells isolated at different developmental stages in a rat model.

Fetal cartilage-derived progenitor cells (FCPCs) could be a useful cell source in cell-based therapies for cartilage disorders. However, their characteristics can vary depending on the developmental stages. The aim of this study was to compare the characteristics of rat FCPCs from the hind limb on embryonic day 14 (E14), E16 and E20 regarding proliferation, pluripotency, and differentiation. Morphologically, rat fetal cartilage tissue showed an increase in cartilaginous differentiation features (Safranin-O, type II collagen) and decrease in pluripotency marker (Sox2) in the order of E14, E16 and E20. E14 FCPCs showed significantly higher doubling time compared to E16 and E20 FCPCs. While the E14 FCPCs expressed pluripotent genes (Sox2, Oct4, Nanog), the E16 and E20 FCPCs expressed chondrogenic markers (Sox9, Col2a1, Acan). E20 FCPCs showed the highest ability to both chondrogenic and adipogenic differentiation and E14 FCPCs showed relatively better activity in osteogenic differentiation. Further analysis showed that E20 FCPCs expressed both adipogenic (C/ebpß) and osteogenic (Runx2, Sp7, Taz) transcription factors as well as chondrogenic transcription factors. Our results show an inverse relationship overall between the expression of pluripotency genes and that of chondrogenic and lineage-specific genes in FCPCs under development. Due to its exceptional proliferation and chondrogenic differentiation ability, fetal cells from epiphyseal cartilage (E20 in rats) may be a suitable cell source for cartilage regeneration.

Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.

The prospective isolation of purified stem cell populations has dramatically altered the field of stem cell biology, and it has been a major focus of research across tissues in different organisms. Muscle stem cells (MuSCs) are now among the most intensely studied stem cell populations in mammalian systems, and the prospective isolation of these cells has allowed cellular and molecular characterizations that were not dreamed of a decade ago. In this protocol, we describe how to isolate MuSCs from limb muscles of adult mice by fluorescence-activated cell sorting (FACS). We provide a detailed description of the physical and enzymatic dissociation of mononucleated cells from limb muscles, a procedure that is essential in order to maximize cell yield. We also describe a FACS-based method that is used subsequently to obtain highly pure populations of either quiescent or activated MuSCs (VCAM(+)CD31(-)CD45(-)Sca1(-)). The isolation process takes ∼5-6 h to complete. The protocol also allows for the isolation of endothelial cells, hematopoietic cells and mesenchymal stem cells from muscle tissue.

Slow- and fast-twitch hindlimb skeletal muscle phenotypes 12 wk after ⠚ nephrectomy in Wistar rats of both sexes.

This study describes fiber-type adaptations in hindlimb muscles, the interaction of sex, and the role of hypoxia on this response in 12-wk ⅚ nephrectomized rats (Nx). Contractile, metabolic, and morphological features of muscle fiber types were assessed in the slow-twitch soleus and the fast-twitch tibialis cranialis muscles of Nx rats, and compared with sham-operated controls. Rats of both sexes were considered in both groups. A slow-to-fast fiber-type transformation occurred in the tibialis cranialis of Nx rats, particularly in males. This adaptation was accomplished by impaired oxidative capacity and capillarity, increased glycolytic capacity, and no changes in size and nuclear density of muscle fiber types. An oxidative-to-glycolytic metabolic transformation was also found in the soleus muscle of Nx rats. However, a modest fast-to-slow fiber-type transformation, fiber hypertrophy, and nuclear proliferation were observed in soleus muscle fibers of male, but not of female, Nx rats. Serum testosterone levels decreased by 50% in male but not in female Nx rats. Hypoxia-inducible factor-1α protein level decreased by 42% in the tibialis cranialis muscle of male Nx rats. These data demonstrate that 12 wk of Nx induces a muscle-specific adaptive response in which myofibers do not change (or enlarge minimally) in size and nuclear density, but acquire markedly different contractile and metabolic characteristics, which are accompanied by capillary rarefaction. Muscle function and sex play relevant roles in these adaptations.

Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells.

Spinal motor neurons (MNs) control diverse motor tasks including respiration, posture and locomotion that are disrupted by neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Methods directing MN differentiation from stem cells have been developed to enable disease modelling in vitro. However, most protocols produce only a limited subset of endogenous MN subtypes. Here we demonstrate that limb-innervating lateral motor column (LMC) MNs can be efficiently generated from mouse and human embryonic stem cells through manipulation of the transcription factor Foxp1. Foxp1-programmed MNs exhibit features of medial and lateral LMC MNs including expression of specific motor pool markers and axon guidance receptors. Importantly, they preferentially project axons towards limb muscle explants in vitro and distal limb muscles in vivo upon transplantation-hallmarks of bona fide LMC MNs. These results present an effective approach for generating specific MN populations from stem cells for studying MN development and disease.