Skeletal muscle stem cells differentiated from human-induced pluripotent stem cells (hiPSCs) serve as a uniquely promising model system for investigating human myogenesis and disease pathogenesis, and for the development of gene editing and regenerative stem cell therapies. Here, we present an effective and reproducible transgene-free protocol for derivation of human skeletal muscle stem cells, iMyoblasts, from hiPSCs. Our two-step protocol consists of 1) small molecule-based differentiation of hiPSCs into myocytes, and 2) stimulation of differentiated myocytes with growth factor-rich medium to activate the proliferation of undifferentiated reserve cells, for expansion and cell line establishment. iMyoblasts are PAX3 + /MyoD1 + myogenic stem cells with dual potential to undergo muscle differentiation and to self-renew as a regenerative cell population for muscle regeneration both ex vivo and in vivo . The simplicity and robustness of iMyoblast generation and expansion have enabled their application to model the molecular pathogenesis of Facioscapulohumeral Muscular Dystrophy and Limb-Girdle Muscular Dystrophies, to both ex vivo and in vivo muscle xenografts, and to respond efficiently to gene editing, enabling the co-development of gene correction and stem cell regenerative therapeutic technologies for the treatment of muscular dystrophies and muscle injury. Graphical abstract.

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for > 12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.

OBJECTIVE: Proinflammatory molecules promote osteoclast-mediated bone erosion by up-regulating local RANKL production. However, recent evidence suggests that combinations of cytokines, such as tumor necrosis factor (TNF) plus interleukin-6 (IL-6), induce RANKL-independent osteoclastogenesis. The purpose of this study was to better understand TNF/IL-6-induced osteoclast formation and to determine whether RANK is absolutely required for osteoclastogenesis and bone erosion in murine inflammatory arthritis. METHODS: Myeloid precursors from wild-type (WT) mice or mice with either germline or conditional deletion of Rank, Nfatc1, Dap12, or Fcrg were treated with either RANKL or TNF plus IL-6. Osteoprotegerin, anti-IL-6 receptor (anti-IL-6R), and hydroxyurea were used to block RANKL, the IL-6R, and cell proliferation, respectively. Clinical scoring, histologic assessment, micro-computed tomography, and quantitative polymerase chain reaction (qPCR) were used to evaluate K/BxN serum-transfer arthritis in WT and RANK-deleted mice. Loss of Rank was verified by qPCR and by osteoclast cultures. RESULTS: TNF/IL-6 generated osteoclasts in vitro that resorbed mineralized tissue through a pathway dependent on IL-6R, NFATc1, DNAX-activation protein 12, and cell proliferation, but independent of RANKL or RANK. Bone erosion and osteoclast formation were reduced, but not absent, in arthritic mice with inducible deficiency of RANK. TNF/IL-6, but not RANKL, induced osteoclast formation in bone marrow and synovial cultures from animals deficient in Rank. Multiple IL-6 family members (IL-6, leukemia inhibitory factor, oncostatin M) were up-regulated in the synovium of arthritic mice. CONCLUSION: The persistence of bone erosion and synovial osteoclasts in Rank-deficient mice, and the ability of TNF/IL-6 to induce osteoclastogenesis, suggest that more than one cytokine pathway exists to generate these bone-resorbing cells in inflamed joints.

Angiogenic biomarkers, including soluble fms-like tyrosine kinase 1 (sFlt1), are thought to be predictors of preeclampsia onset; however, improvement is needed before a widespread diagnostic test can be utilized. Here we describe the development and use of diagnostic monoclonal antibodies specific to the two main splice variants of sFlt1, sFlt1-1 and sFlt1-14. These antibodies were selected for their sensitivity and specificity to their respective sFlt1 isoform in a capture ELISA format. Data from this pilot study suggest that sFlt1-1 may be more predictive of preeclampsia than total sFlt1. It may be possible to improve current diagnostic platforms if more specific antibodies are utilized.

OBJECTIVE: Chronic hypertension, pregestational diabetes mellitus, history of prior preeclampsia and obese nulliparity are maternal conditions associated with increased preeclampsia risk. Whether altered maternal angiogenic factor levels allow for prediction of pending disease is unclear. Our objective was to evaluate angiogenic factors for early preeclampsia prediction in high-risk women. METHODS: Serial serum specimens were collected from 157 women at high preeclampsia risk and 50 low-risk controls between 23 and 36 weeks gestation in 3 windows (23-27.6, 28-31.6, and 32-35.6 weeks) in a two-center observational cohort. Soluble fms-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF) and soluble endoglin (sEng) were measured by ELISA. RESULTS: Multivariate parsimonious logistic regression analyses using backward elimination for prediction of early-preeclampsia (diagnosed < 34 weeks) found the best-fitting model included the predictors (1) sFlt1 measured in the second window (28-31.6 weeks) with AUC 0.85, sensitivity 67% and specificity 96% and (2) sFlt1 measured in the first window (23-27.6 weeks) and sEng change between first and second window with AUC 0.91, sensitivity 86% and specificity 96%. CONCLUSIONS: Two-stage sampling screening protocol utilizing sFlt1 and sEng is promising for prediction of preeclampsia diagnosed before 34 weeks. Larger studies are needed to confirm these findings.

OBJECTIVE: Several conditions are associated with increased preeclampsia (PE) risk. Whether altered maternal angiogenic factor levels contribute to risk in these conditions is unknown. Our objective was to compare angiogenic biomarker patterns in high-risk pregnancies and low-risk controls. STUDY DESIGN: We conducted a planned secondary analysis of a 2-center observational study of angiogenic biomarkers in high-risk women. A total of 156 pregnant women with a PE risk factor and 59 low-risk controls were studied. Serial maternal serum samples were collected during 3 gestational windows: 23-27 weeks, 28-31 weeks, and 32-35 weeks. Soluble fms-like tyrosine kinase 1 (sFlt1), soluble endoglin (sEng), and placental growth factor (PlGF) were measured by enzyme-linked immunosorbent assay. Geometric mean angiogenic biomarker levels and angiogenic ratio (sFlt1 + sEng):PlGF were compared with low-risk controls for each risk group, at each gestational window. RESULTS: Gestational biomarker patterns differed in PE risk groups as compared with low-risk controls. Women with multiple gestations had markedly higher sFlt1 and sEng at all gestational windows. Women with prior PE had higher sFlt1 and angiogenic ratio, and lower PlGF, from 28 weeks onward. Women with chronic hypertension had significantly higher angiogenic ratio for all 3 gestational windows, but differences disappeared when women with PE were excluded. Obese and nulliparous women had significantly lower PlGF, but no differences in the angiogenic ratio. CONCLUSION: High-risk groups have altered angiogenic biomarker patterns compared with controls, suggesting that altered production or metabolism of these factors may contribute to PE risk, particularly in women with multiple gestations and prior PE.

OBJECTIVE: To determine if maternal serum angiogenic factors predict maternal and neonatal complications in women presenting to an acute care setting with suspected preeclampsia. STUDY DESIGN: Maternal serum samples were prospectively collected from women with suspected preeclampsia at the time of initial presentation to hospital triage with signs or symptoms of preeclampsia. Soluble fms-like tyrosine kinase-1 (sFlt1), placental growth factor (PlGF), and soluble endoglin (sEng) were measured by ELISA. The primary outcome was a composite of maternal and neonatal complications. RESULTS: Of 276 women with suspected preeclampsia, 78 developed maternal or neonatal complications. Among women presenting prior to 37 weeks gestation, sFlt1, PlGF, and sEng were significantly different in women who developed maternal and neonatal complications as compared to women without complications. Higher levels of sFlt1, sEng, and the sFlt1:PlGF ratio were associated with an increased odds of complications among women presenting prior to 37 weeks. A multivariable model combining the sFlt1:PlGF ratio with clinical variables was more predictive of complications (AUC 0.91, 95% CI 0.85-0.97) than a model using clinical variables alone (AUC 0.82, 95% CI 0.79-0.90). CONCLUSION: Angiogenic biomarkers associate with maternal and neonatal complications in women with suspected preeclampsia, and may be useful for risk stratification.

Trefoil family factor 2 (TFF2), also known as spasmolytic peptide, is a low-molecular-weight protein that is upregulated in gastric tissues infected with Helicobacter or having other inflammatory conditions, but a precise function is yet to be elucidated. The role of TFF2 in the development of gastritis, colitis, and inflammatory cytokine responses was examined both in vivo and in vitro using wild-type and TFF2 knockout mice. TFF2 knockout and wild-type mice were infected with Helicobacter felis (H. felis) to induce gastritis. Colitis was induced in TFF2 knockout and wild-type mice by administering dextran sodium sulfate (DSS) in drinking water. Histopathology, clinical disease (colitis), and antibody levels (H. felis) were examined. TFF2 expression in tissues was determined by reverse transcriptase PCR, and the inflammatory and proliferative responses of TFF2-expressing macrophages and spleen cells were examined by cytokine enzyme-linked immunosorbent assay, thymidine incorporation, and gene array studies. TFF2 knockout mice have increased susceptibility to H. felis-induced gastritis, with enhanced gastric inflammation. They were also more susceptible to DSS-induced colitis, with prolonged colonic hemorrhage and persistent weight loss. Remarkably, TFF2 expression was not limited to the gastrointestinal tract, as suggested in previous studies, but was also present in macrophages and lymphocytes. The inflammatory and proliferative responses of these immune cell types were dysregulated in TFF2 knockout mice. TFF2-/- cells were hyperresponsive to interleukin 1 beta stimulation but showed normal responses to lipopolysaccharide, suggesting a specific role for TFF2 in interleukin 1 receptor but not Toll-like receptor 4 signaling via their Toll-interleukin 1 resistance domains. TFF2-/- lymphocytes also produced higher levels of interleukin 2 than wild-type cells. Thus, TFF2 was expressed in the gastrointestinal cells and in immune cells and was a negative regulator of gastrointestinal inflammation and immune cell cytokine responses. Our studies suggest that TFF2 not only controls gastrointestinal repair but also regulates mononuclear cell inflammatory responses.