GFAP-expressing cells in the adult hypothalamus can generate multiple neural cell lineages in vitro.

Adult neural stem cells (NSCs) located in the two canonical neurogenic niches, the subventricular zone (SVZ) and the subgranular zone (SGZ), express the glial fibrillary acidic protein (GFAP). Recently, proliferative activity has been described in the hypothalamus although the characterization of hypothalamic neural stem/progenitor cells (NSPCs) is still uncertain. We therefore investigated whether hypothalamic GFAP-positive cells, as in the SVZ and SGZ, also have neurogenic potential. We used a transgenic mouse line expressing green fluorescent protein (GFP) under the control of the GFAP promoter. GFAP-GFP expressing cells are localized in the ependymal layer as well as in the parenchyma of the mediobasal hypothalamus (MBH) and express Sox2, a marker for NSCs. Interestingly, no sexual dimorphism was observed in the numbers of GFP + and GFP-Sox2 + cells. After cells sorting, these cells were able to generate neurospheres in vitro and give rise to neurons, astrocytes and oligodendrocytes. Taken together, these results show that hypothalamic GFAP-expressing cells form a population of NSPCs.

Gadolinium-Based Contrast Agents and Free Gadolinium Inhibit Differentiation and Activity of Bone Cell Lineages.

OBJECTIVES: Administration of gadolinium-based contrast agents (GBCA) in magnetic resonance imaging results in the long-term retention of gadolinium (Gd) in tissues and organs, including the bone, and may affect their function and metabolism. This study aims to investigate the effects of Gd and GBCA on the proliferation/survival, differentiation, and function of bone cell lineages. MATERIALS AND METHODS: Primary murine osteoblasts (OB) and osteoclast progenitor cells (OPC) isolated from C57BL/6J mice were used to test the effects of Gd 3+ (12.5-100 µM) and GBCA (100-2000 µM). Cultures were supplemented with the nonionic linear Gd-DTPA-BMA (gadodiamide), ionic linear Gd-DTPA (gadopentetic acid), and macrocyclic Gd-DOTA (gadoteric acid). Cell viability and differentiation were analyzed on days 4-6 of the culture. To assess the resorptive activity of osteoclasts, the cells were grown in OPC cultures and were seeded onto layers of amorphous calcium phosphate with incorporated Gd. RESULTS: Gd 3+ did not affect OB viability, but differentiation was reduced dose-dependently up to 72.4% ± 6.2%-73.0% ± 13.2% (average ± SD) at 100 µM Gd 3+ on days 4-6 of culture as compared with unexposed controls ( P < 0.001). Exposure to GBCA had minor effects on OB viability with a dose-dependent reduction up to 23.3% ± 10.2% for Gd-DTPA-BMA at 2000 µM on day 5 ( P < 0.001). In contrast, all 3 GBCA caused a dose-dependent reduction of differentiation up to 88.3% ± 5.2% for Gd-DTPA-BMA, 49.8% ± 16.0% for Gd-DTPA, and 23.1% ± 8.7% for Gd-DOTA at 2000 µM on day 5 ( P < 0.001). In cultures of OPC, cell viability was not affected by Gd 3+ , whereas differentiation was decreased by 45.3% ± 9.8%-48.5% ± 15.8% at 100 µM Gd 3+ on days 4-6 ( P < 0.05). Exposure of OPC to GBCA resulted in a dose-dependent increase in cell viability of up to 34.1% ± 11.4% at 2000 µM on day 5 of culture ( P < 0.001). However, differentiation of OPC cultures was reduced on day 5 by 24.2% ± 9.4% for Gd-DTPA-BMA, 47.1% ± 14.0% for Gd-DTPA, and 38.2% ± 10.0% for Gd-DOTA ( P < 0.001). The dissolution of amorphous calcium phosphate by mature osteoclasts was reduced by 36.3% ± 5.3% upon incorporation of 4.3% Gd/Ca wt/wt ( P < 0.001). CONCLUSIONS: Gadolinium and GBCA inhibit differentiation and activity of bone cell lineages in vitro. Thus, Gd retention in bone tissue could potentially impair the physiological regulation of bone turnover on a cellular level, leading to pathological changes in bone metabolism.

Deficiency in the omega-3 lysolipid transporter Mfsd2a leads to aberrant oligodendrocyte lineage development and hypomyelination.

Patients with autosomal recessive microcephaly 15 caused by deficiency in the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain-containing 2a (Mfsd2a) present with both microcephaly and hypomyelination, suggesting an important role for LPC uptake by oligodendrocytes in the process of myelination. Here we demonstrate that Mfsd2a is specifically expressed in oligodendrocyte precursor cells (OPCs) and is critical for oligodendrocyte development. Single-cell sequencing of the oligodendrocyte lineage revealed that OPCs from OPC-specific Mfsd2a-KO mice (2aOKO mice) underwent precocious differentiation into immature oligodendrocytes and impaired maturation into myelinating oligodendrocytes, correlating with postnatal brain hypomyelination. 2aOKO mice did not exhibit microcephaly, a finding consistent with the notion that microcephaly is the consequence of an absence of LPC uptake at the blood-brain barrier rather than a deficiency in OPCs. Lipidomic analysis showed that OPCs and iOLs from 2aOKO mice had significantly decreased levels of phospholipids containing omega-3 fatty acids, with a corresponding increase in unsaturated fatty acids, the latter being products of de novo synthesis governed by Srebp-1. RNA-Seq indicated activation of the Srebp-1 pathway and defective expression of regulators of oligodendrocyte development. Taken together, these findings indicate that the transport of LPCs by Mfsd2a in OPCs is important for maintaining OPC state to regulate postnatal brain myelination.

Hierarchical deployment of Tbx3 dictates the identity of hypothalamic KNDy neurons to control puberty onset.

The neuroendocrine system consists of a heterogeneous collection of neuropeptidergic neurons in the brain, among which hypothalamic KNDy neurons represent an indispensable cell subtype controlling puberty onset. Although neural progenitors and neuronal precursors along the cell lineage hierarchy adopt a cascade diversification strategy to generate hypothalamic neuronal heterogeneity, the cellular logic operating within the lineage to specify a subtype of neuroendocrine neurons remains unclear. As human genetic studies have recently established a link between TBX3 mutations and delayed puberty onset, we systematically studied Tbx3-derived neuronal lineage and Tbx3-dependent neuronal specification and found that Tbx3 hierarchically established and maintained the identity of KNDy neurons for triggering puberty. Apart from the well-established lineage-dependent fate determination, we uncovered rules of interlineage interaction and intralineage retention operating through neuronal differentiation in the absence of Tbx3. Moreover, we revealed that human TBX3 mutations disturbed the phase separation of encoded proteins and impaired transcriptional regulation of key neuropeptides, providing a pathological mechanism underlying TBX3-associated puberty disorders.

Systems-level identification of key transcription factors in immune cell specification.

Transcription factors (TFs) are crucial for regulating cell differentiation during the development of the immune system. However, the key TFs for orchestrating the specification of distinct immune cells are not fully understood. Here, we integrated the transcriptomic and epigenomic measurements in 73 mouse and 61 human primary cell types, respectively, that span the immune cell differentiation pathways. We constructed the cell-type-specific transcriptional regulatory network and assessed the global importance of TFs based on the Taiji framework, which is a method we have previously developed that can infer the global impact of TFs using integrated transcriptomic and epigenetic data. Integrative analysis across cell types revealed putative driver TFs in cell lineage-specific differentiation in both mouse and human systems. We have also identified TF combinations that play important roles in specific developmental stages. Furthermore, we validated the functions of predicted novel TFs in murine CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Thus, we have developed a bioinformatic approach that provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions.

H3K27 methylation regulates the fate of two cell lineages in male gametophytes.

During angiosperm male gametogenesis, microspores divide to produce a vegetative cell (VC) and a male germline (MG), each with distinct cell fates. The mechanism underlying determination of the MG cell/VC fate remains an important area of research, with many unanswered questions. Here, we report that H3K27me3 is essential for VC fate commitment in male Arabidopsis thaliana gametophytes; H3K27me3 erasure contributes to MG cell fate initiation. VC-targeted H3K27me3 erasure disturbed VC development and shifted the VC fate toward a gamete destination, which suggests that MG cells require H3K27me3 erasure to trigger gamete cell fate. Multi-omics and cytological analyses confirmed the occurrence of extensive cell identity transition due to H3K27me3 erasure. Therefore, we experimentally confirmed that MG cell/VC fate is epigenetically regulated. H3K27 methylation plays a critical role in guiding MG cell/VC fate determination for pollen fertility in Arabidopsis. Our work also provides evidence for two previous hypotheses: the germline cell fate is specified by the differential distribution of unknown determinants and VC maintains the default microspore program (i.e. the H3K27me3 setting) while MG requires reprogramming.

Imipridones affect tumor bioenergetics and promote cell lineage differentiation in diffuse midline gliomas.

BACKGROUND: Pediatric diffuse midline gliomas (DMGs) are incurable childhood cancers. The imipridone ONC201 has shown early clinical efficacy in a subset of DMGs. However, the anticancer mechanisms of ONC201 and its derivative ONC206 have not been fully described in DMGs. METHODS: DMG models including primary human in vitro (n = 18) and in vivo (murine and zebrafish) models, and patient (n = 20) frozen and FFPE specimens were used. Drug-target engagement was evaluated using in silico ChemPLP and in vitro thermal shift assay. Drug toxicity and neurotoxicity were assessed in zebrafish models. Seahorse XF Cell Mito Stress Test, MitoSOX and TMRM assays, and electron microscopy imaging were used to assess metabolic signatures. Cell lineage differentiation and drug-altered pathways were defined using bulk and single-cell RNA-seq. RESULTS: ONC201 and ONC206 reduce viability of DMG cells in nM concentrations and extend survival of DMG PDX models (ONC201: 117 days, P = .01; ONC206: 113 days, P = .001). ONC206 is 10X more potent than ONC201 in vitro and combination treatment was the most efficacious at prolonging survival in vivo (125 days, P = .02). Thermal shift assay confirmed that both drugs bind to ClpP, with ONC206 exhibiting a higher binding affinity as assessed by in silico ChemPLP. ClpP activation by both drugs results in impaired tumor cell metabolism, mitochondrial damage, ROS production, activation of integrative stress response (ISR), and apoptosis in vitro and in vivo. Strikingly, imipridone treatment triggered a lineage shift from a proliferative, oligodendrocyte precursor-like state to a mature, astrocyte-like state. CONCLUSION: Targeting mitochondrial metabolism and ISR activation effectively impairs DMG tumorigenicity. These results supported the initiation of two pediatric clinical trials (NCT05009992, NCT04732065).

Photobiomodulation has rejuvenating effects on aged bone marrow mesenchymal stem cells.

The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm2 is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.

An Intermediate Concentration of Calcium with Antioxidant Supplement in Culture Medium Enhances Proliferation and Decreases the Aging of Bone Marrow Mesenchymal Stem Cells.

Human bone marrow stem cells (HBMSCs) are isolated from the bone marrow. Stem cells can self-renew and differentiate into various types of cells. They are able to regenerate kinds of tissue that are potentially used for tissue engineering. To maintain and expand these cells under culture conditions is difficult-they are easily triggered for differentiation or death. In this study, we describe a new culture formula to culture isolated HBMSCs. This new formula was modified from NCDB 153, a medium with low calcium, supplied with 5% FBS, extra growth factor added to it, and supplemented with N-acetyl-L-cysteine and L-ascorbic acid-2-phosphate to maintain the cells in a steady stage. The cells retain these characteristics as primarily isolated HBMSCs. Moreover, our new formula keeps HBMSCs with high proliferation rate and multiple linage differentiation ability, such as osteoblastogenesis, chondrogenesis, and adipogenesis. It also retains HBMSCs with stable chromosome, DNA, telomere length, and telomerase activity, even after long-term culture. Senescence can be minimized under this new formulation and carcinogenesis of stem cells can also be prevented. These modifications greatly enhance the survival rate, growth rate, and basal characteristics of isolated HBMSCs, which will be very helpful in stem cell research.

Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL).

USP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.

Dual midbrain and forebrain origins of thalamic inhibitory interneurons.

The ubiquitous presence of inhibitory interneurons in the thalamus of primates contrasts with the sparsity of interneurons reported in mice. Here, we identify a larger than expected complexity and distribution of interneurons across the mouse thalamus, where all thalamic interneurons can be traced back to two developmental programmes: one specified in the midbrain and the other in the forebrain. Interneurons migrate to functionally distinct thalamocortical nuclei depending on their origin: the abundant, midbrain-derived class populates the first and higher order sensory thalamus while the rarer, forebrain-generated class is restricted to some higher order associative regions. We also observe that markers for the midbrain-born class are abundantly expressed throughout the thalamus of the New World monkey marmoset. These data therefore reveal that, despite the broad variability in interneuron density across mammalian species, the blueprint of the ontogenetic organisation of thalamic interneurons of larger-brained mammals exists and can be studied in mice.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

A novel telencephalon-opto-hypothalamic morphogenetic domain coexpressing Foxg1 and Otp produces most of the glutamatergic neurons of the medial extended amygdala.

Deficits in social cognition and behavior are a hallmark of many psychiatric disorders. The medial extended amygdala, including the medial amygdala and the medial bed nucleus of the stria terminalis, is a key component of functional networks involved in sociality. However, this nuclear complex is highly heterogeneous and contains numerous GABAergic and glutamatergic neuron subpopulations. Deciphering the connections of different neurons is essential in order to understand how this structure regulates different aspects of sociality, and it is necessary to evaluate their differential implication in distinct mental disorders. Developmental studies in different vertebrates are offering new venues to understand neuronal diversity of the medial extended amygdala and are helping to establish a relation between the embryonic origin and molecular signature of distinct neurons with the functional subcircuits in which they are engaged. These studies have provided many details on the distinct GABAergic neurons of the medial extended amygdala, but information on the glutamatergic neurons is still scarce. Using an Otp-eGFP transgenic mouse and multiple fluorescent labeling, we show that most glutamatergic neurons of the medial extended amygdala originate in a distinct telencephalon-opto-hypothalamic embryonic domain (TOH), located at the transition between telencephalon and hypothalamus, which produces Otp-lineage neurons expressing the telencephalic marker Foxg1 but not Nkx2.1 during development. These glutamatergic cells include a subpopulation of projection neurons of the medial amygdala, which activation has been previously shown to promote autistic-like behavior. Our data open new venues for studying the implication of this neuron subtype in neurodevelopmental disorders producing social deficits.

T Helper Cell Lineage-Defining Transcription Factors: Potent Targets for Specific GVHD Therapy?

Allogenic hematopoietic stem cell transplantation (allo-HSCT) represents a potent and potentially curative treatment for many hematopoietic malignancies and hematologic disorders in adults and children. The donor-derived immunity, elicited by the stem cell transplant, can prevent disease relapse but is also responsible for the induction of graft-versus-host disease (GVHD). The pathophysiology of acute GVHD is not completely understood yet. In general, acute GVHD is driven by the inflammatory and cytotoxic effect of alloreactive donor T cells. Since several experimental approaches indicate that CD4 T cells play an important role in initiation and progression of acute GVHD, the contribution of the different CD4 T helper (Th) cell subtypes in the pathomechanism and regulation of the disease is a central point of current research. Th lineages derive from naïve CD4 T cell progenitors and lineage commitment is initiated by the surrounding cytokine milieu and subsequent changes in the transcription factor (TF) profile. Each T cell subtype has its own effector characteristics, immunologic function, and lineage specific cytokine profile, leading to the association with different immune responses and diseases. Acute GVHD is thought to be mainly driven by the Th1/Th17 axis, whereas Treg cells are attributed to attenuate GVHD effects. As the differentiation of each Th subset highly depends on the specific composition of activating and repressing TFs, these present a potent target to alter the Th cell landscape towards a GVHD-ameliorating direction, e.g. by inhibiting Th1 and Th17 differentiation. The finding, that targeting of Th1 and Th17 differentiation appears more effective for GVHD-prevention than a strategy to inhibit Th1 and Th17 cytokines supports this concept. In this review, we shed light on the current advances of potent TF inhibitors to alter Th cell differentiation and consecutively attenuate GVHD. We will focus especially on preclinical studies and outcomes of TF inhibition in murine GVHD models. Finally, we will point out the possible impact of a Th cell subset-specific immune modulation in context of GVHD.

Comparison of responses of melanocyte lineages from p75(+) and p75(-) human scalp-derived neural crest stem cells under phototherapy.

Phototherapy is an effective therapeutic option in the treatment of vitiligo; however, responses varied among the different types. The underlying mechanism has scarcely been investigated. To investigate and compare the effects of phototherapy on the mutation of melanocyte lineage differentiated from human scalp-derived neural crest stem cells (HS-NCSCs) with p75 neurotrophin receptor expression positive and p75 neurotrophin receptor expression negative group in vitro, the HS-NCSCs were isolated from fetal scalp tissue, which is identified by immunofluorescent staining. The p75(+) and p75(-) cells from HS-NCSCs were isolated by magnetic cell sorting, respectively. The embryonic neural crest stem cell biomarkers were detected by RT-PCR. Narrow-band UVB (NB-UVB) was used to irradiate the cells. Cell proliferation was evaluated by cell count. Tyrosinase, Tyrp1, and Tyrp2 gene expression were measured by quantitative RT-PCR. Tyrosinase and GRCR protein levels were investigated by Western blot analysis. The electrophoretic strip showed that Sox2, Oct4, Sox10, and Nestin of p75(+) HS-NCSCs were brighter than the p75(-) HS-NCSCs. After the same dose radiation with NB-UVB, the cell proliferation of p75(+) group showed less inhibitory rate compared with the p75(-) HS-NCSCs. The tyrosinase mRNA and protein expression of differentiated melanocytes increased significantly in the group of p75(+) HS-NCSCs compared with the p75(-) group. The melanocytic mutation of p75(+) HS-NCSCs increased significantly compared with the p75(-) HS-NCSCs under NB-UVB, which indicated there were more melanocyte precursors in the differentiated cells from p75(+) HS-NCSCs. This may provide new insights for the different repigmentation efficacy of segmental and non-segmental vitiligo.

DNA methylation dynamics of sperm cell lineage development in tomato.

During the sexual reproduction of higher plants, DNA methylation and transcription are broadly changed to reshape a microspore into two sperm cells (SCs) and a vegetative cell (VC). However, when and how the DNA methylation of SCs is established remains not fully understood. Here we investigate the DNA methylation (5 mC) dynamics of SC lineage and the VC in tomato using whole-genome bisulfite sequencing. We find the asymmetric division of the microspore gives its two daughter cells differential methylome. Compared with the generative cell (GC), the VC is hypomethylated at CG sites while hypermethylated at CHG and CHH sites, with the majority of differentially methylation regions targeted to transposable elements (TEs). SCs have a nearly identical DNA methylome to the GC, suggesting that the methylation landscape in SCs may be pre-established following the asymmetric division or inherited from the GC. The random forest classifier for predicting gene and TE expression shows that methylation within the gene body is a more powerful predictor for gene expression. Among all tested samples, gene and TE expression in the microspore may be more predictable by DNA methylation. Our results depict an intact DNA methylome landscape of SC lineage in higher plants, and reveal that the impact of DNA methylation on transcription is variant in different cell types.

Effect of Anadenanthera colubrina protease inhibitors as an antiinflamatory mediator.

This work aimed to obtain and characterize protease inhibitors from A. colubrina leaves and evaluate their potential as inflammatory mediator and cell viability. The protein extract was analyzed and characterized by SDS-PAGE, RP-HPLC-PDA, MALDI-TOF/MS and Zeta potential. Bioassays were conducted in order to evaluate the cell viability in RAW 264.7, in vitro (NO and TNF-α production inhibition) and in vivo anti-inflammatory potential, inhibition rate of trypsin and hemagglutination activity from protein extract. The results revealed the presence of bands at 14, 21 and 30 kDa in SDS-PAGE, the RP-HPLC-PDA analysis showed peaks at 12, 13, 28 and 40 minutes and MALDI-TOF/MS showed peaks with 3.4, 4.7, 5.6, 9.4 and 11.2 kDa. The protein extracts presented enzymatic activity inhibition of trypsin (IC50 59.2 µgmL-1), did not show any cytotoxicity to RAW264.7 cells, hemagglutination 8HU and insignificant reduction in NO and TNF-α production and reduced anti-inflammatory potential in vivo compared to dexamethasone.

The Vitamin D Receptor in Osteoblast-Lineage Cells Is Essential for the Proresorptive Activity of 1α,25(OH)2D3 In Vivo.

We previously reported that daily administration of a pharmacological dose of eldecalcitol, an analog of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], increased bone mass by suppressing bone resorption. These antiresorptive effects were found to be mediated by the vitamin D receptor (VDR) in osteoblast-lineage cells. Using osteoblast-lineage-specific VDR conditional knockout (Ob-VDR-cKO) mice, we examined whether proresorptive activity induced by the high-dose 1α,25(OH)2D3 was also mediated by VDR in osteoblast-lineage cells. Administration of 1α,25(OH)2D3 (5 µg/kg body weight/day) to wild-type mice for 4 days increased the number of osteoclasts in bone and serum concentrations of C-terminal crosslinked telopeptide of type I collagen (CTX-I, a bone resorption marker). The stimulation of bone resorption was concomitant with the increase in serum calcium (Ca) and fibroblast growth factor 23 (FGF23) levels, and decrease in body weight. This suggests that a toxic dose of 1α,25(OH)2D3 can induce bone resorption and hypercalcemia. In contrast, pretreatment of wild-type mice with neutralizing anti-receptor activator of NF-κB ligand (RANKL) antibody inhibited the 1α,25(OH)2D3-induced increase of osteoclast numbers in bone, and increase of CTX-I, Ca, and FGF23 levels in serum. The pretreatment with anti-RANKL antibody also inhibited the 1α,25(OH)2D3-induced decrease in body weight. Consistent with observations in mice conditioned with anti-RANKL antibody, the high-dose administration of 1α,25(OH)2D3 to Ob-VDR-cKO mice failed to significantly increase bone osteoclast numbers, serum CTX-I, Ca, or FGF23 levels, and failed to reduce the body weight. Taken together, this study demonstrated that the proresorptive, hypercalcemic, and toxic actions of high-dose 1α,25(OH)2D3 are mediated by VDR in osteoblast-lineage cells.

Conditional knockout of leptin receptor in neural stem cells leads to obesity in mice and affects neuronal differentiation in the hypothalamus early after birth.

Leptin, secreted by peripheral adipocytes, binds the leptin receptor (Lepr) in the hypothalamus, thereby contributing to the regulation of satiety and body weight. Lepr is expressed in the embryonic brain as early as embryonic day 12.5. However, the function of Lepr in neural precursor cells in the brain has not been resolved. To address this issue, we crossed the Leprflox/flox mice with each of Shh-Cre mice (Shh, sonic hedgehog) and Nestin (Nes)-Cre mice. We found that deletion of Lepr specifically in nestin-expressing cells led to extreme obesity, but the conditional null of Lepr in Shh-expressing cells had no obvious phenotype. Moreover, the level of leptin-activated pSTAT3 decreased in the anterior and central subregions of the arcuate hypothalamus of Shh-Cre; Leprflox/flox mice compared with the controls. By contrast, in Nes-Cre; Leprflox/flox mice, the level of leptin-activated pSTAT3 decreased in all subregions including the anterior, central, and posterior arcuate hypothalamus as well as the dorsomedial, ventromedial, and median eminence of the hypothalamus, revealing that the extensive lack of Lepr in the differentiated neurons of the hypothalamus in the conditional null mice. Notably, conditional deletion of Lepr in nestin-expressing cells enhanced the differentiation of neural precursor cells into neurons and oligodendroglia but inhibited differentiation into astrocytes early in postnatal development of hypothalamus. Our results suggest that Lepr expression in neural precursor cells is essential for maintaining normal body weight as well as the differentiation of neural precursor cells to the neural/glial fate in the hypothalamus shortly after birth.

Human Cardiac Mesenchymal Stem Cells Remodel in Disease and Can Regulate Arrhythmia Substrates.

BACKGROUND: The mesenchymal stem cell (MSC), known to remodel in disease and have an extensive secretome, has recently been isolated from the human heart. However, the effects of normal and diseased cardiac MSCs on myocyte electrophysiology remain unclear. We hypothesize that in disease the inflammatory secretome of cardiac human MSCs (hMSCs) remodels and can regulate arrhythmia substrates. METHODS: hMSCs were isolated from patients with or without heart failure from tissue attached to extracted device leads and from samples taken from explanted/donor hearts. Failing hMSCs or nonfailing hMSCs were cocultured with normal human cardiac myocytes derived from induced pluripotent stem cells. Using fluorescent indicators, action potential duration, Ca2+ alternans, and spontaneous calcium release (SCR) incidence were determined. RESULTS: Failing and nonfailing hMSCs from both sources exhibited similar trilineage differentiation potential and cell surface marker expression as bone marrow hMSCs. Compared with nonfailing hMSCs, failing hMSCs prolonged action potential duration by 24% (P<0.001, n=15), increased Ca2+ alternans by 300% (P<0.001, n=18), and promoted spontaneous calcium release activity (n=14, P<0.013) in human cardiac myocytes derived from induced pluripotent stem cells. Failing hMSCs exhibited increased secretion of inflammatory cytokines IL (interleukin)-1ß (98%, P<0.0001) and IL-6 (460%, P<0.02) compared with nonfailing hMSCs. IL-1ß or IL-6 in the absence of hMSCs prolonged action potential duration but only IL-6 increased Ca2+ alternans and promoted spontaneous calcium release activity in human cardiac myocytes derived from induced pluripotent stem cells, replicating the effects of failing hMSCs. In contrast, nonfailing hMSCs prevented Ca2+ alternans in human cardiac myocytes derived from induced pluripotent stem cells during oxidative stress. Finally, nonfailing hMSCs exhibited >25× higher secretion of IGF (insulin-like growth factor)-1 compared with failing hMSCs. Importantly, IGF-1 supplementation or anti-IL-6 treatment rescued the arrhythmia substrates induced by failing hMSCs. CONCLUSIONS: We identified device leads as a novel source of cardiac hMSCs. Our findings show that cardiac hMSCs can regulate arrhythmia substrates by remodeling their secretome in disease. Importantly, therapy inhibiting (anti-IL-6) or mimicking (IGF-1) the cardiac hMSC secretome can rescue arrhythmia substrates.