Dysfunctional inflammatory pathways are associated with an increased risk of cancer, including colorectal cancer. We have previously identified and enriched for a self-renewing, colon cancer stem ...cell (CCSC) subpopulation in primary sporadic colorectal cancers (CRC) and a related subpopulation in ulcerative colitis (UC) patients defined by the stem cell marker, aldehyde dehydrogenase (ALDH). Subsequent work demonstrated that CCSC-initiated tumors are dependent on the inflammatory chemokine, CXCL8, a known inducer of tumor proliferation, angiogenesis and invasion. Here, we use RNA interference to target CXCL8 and its receptor, CXCR1, to establish the existence of a functional signaling pathway promoting tumor growth initiated by sporadic and colitis CCSCs. Knocking down either CXCL8 or CXCR1 had a dramatic effect on inhibiting both in vitro proliferation and angiogenesis. Likewise, tumorigenicity was significantly inhibited due to reduced levels of proliferation and angiogenesis. Decreased expression of cycle cell regulators cyclins D1 and B1 along with increased p21 levels suggested that the reduction in tumor growth is due to dysregulation of cell cycle progression. Therapeutically targeting the CXCL8-CXCR1 signaling pathway has the potential to block sustained tumorigenesis by inhibiting both CCSC- and pCCSC-induced proliferation and angiogenesis.
Reports of neural transdifferentiation of mesenchymal stem cells (MSCs) suggest the possibility that these cells may serve as a source for stem cell-based regenerative medicine to treat neurological ...disorders. However, some recent studies controvert previous reports of MSC neurogenecity. In the current study, we evaluate the neural differentiation potential of mouse bone marrow-derived MSCs. Surprisingly, we found that MSCs spontaneously express certain neuronal phenotype markers in culture, in the absence of specialized induction reagents. A previously published neural induction protocol that elevates cytoplasmic cyclic AMP does not upregulate neuron-specific protein expression significantly in MSCs but does significantly increase expression of the astrocyte-specific glial fibrillary acidic protein. Finally, when grafted into the lateral ventricles of neonatal mouse brain, MSCs migrate extensively and differentiate into olfactory bulb granule cells and periventricular astrocytes, without evidence of cell fusion. These results indicate that MSCs may be "primed" toward a neural fate by the constitutive expression of neuronal antigens and that they seem to respond with an appropriate neural pattern of differentiation when exposed to the environment of the developing brain.
Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low ...stoichiometric phosphopeptide. We develop a tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3000 (>9500) phosphopeptides from 1 (10) µg of cell lysate using the label-free method without a spectral library. It also enables precise quantification of ~600 phosphopeptides from 100 sorted cells (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 µm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.
During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and ...escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.
Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be ...particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa−/−) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.
Seventeen international consortia are collaborating on a human reference atlas (HRA), a comprehensive, high-resolution, three-dimensional atlas of all the cells in the healthy human body. ...Laboratories around the world are collecting tissue specimens from donors varying in sex, age, ethnicity, and body mass index. However, harmonizing tissue data across 25 organs and more than 15 bulk and spatial single-cell assay types poses challenges. Here, we present software tools and user interfaces developed to spatially and semantically annotate ("register") and explore the tissue data and the evolving HRA. A key part of these tools is a common coordinate framework, providing standard terminologies and data structures for describing specimen, biological structure, and spatial data linked to existing ontologies. As of April 22, 2022, the "registration" user interface has been used to harmonize and publish data on 5,909 tissue blocks collected by the Human Biomolecular Atlas Program (HuBMAP), the Stimulating Peripheral Activity to Relieve Conditions program (SPARC), the Human Cell Atlas (HCA), the Kidney Precision Medicine Project (KPMP), and the Genotype Tissue Expression project (GTEx). Further, 5,856 tissue sections were derived from 506 HuBMAP tissue blocks. The second "exploration" user interface enables consortia to evaluate data quality, explore tissue data spatially within the context of the HRA, and guide data acquisition. A companion website is at https://cns-iu.github.io/HRA-supporting-information/ .
This report evaluates the spatial profile of blood vessel fragments (BVFs) and CD34+ and CD117+ hematopoietic stem and progenitor cells (HSPCs) in human cancellous bone. Bone specimens were ...sectioned, immunostained (anti-CD34 and anti-CD117), and digitally imaged. Immunoreactive cells and vessels were then optically and morphometrically identified and labeled on the corresponding digital image. The distance of each BVF, or CD34+ or CD117+ HSPC to the nearest trabecular surface was measured and binned in 50-μm increments. The relative concentration of HSPCs and BVFs within cancellous marrow was observed to diminish with increasing distance in the marrow space. On average, 50% of the CD34+ HSPC population, 60% of the CD117+ HSPC population, and 72% of the BVFs were found within 100 μm of the bone surfaces. HSPCs were also found to exist in close proximity to BVFs, which supports the notion of a shared HSPC and vessel spatial niche.
Bone marrow sinusoids maintain homeostasis between developing hematopoietic cells and the circulation, and they provide niches for hematopoietic progenitors. Sinusoids are damaged by chemotherapy and ...radiation. Hematopoietic stem cells (HSCs) have been shown to produce endothelial progenitor cells that contribute to the repair of damaged blood vessels. Because HSCs home to the marrow during bone marrow transplant, these cells may play a role in repair of marrow sinusoids. Here, we explore the role of donor HSCs in the repair of damaged sinusoids following hematopoietic stem cell transplant. We used three methods to test this role: (a) expression of platelet endothelial cell adhesion molecule to identify endothelial progenitors and the presence of the Y chromosome to identify male donor cells in female recipients; (b) presence of the Y chromosome to identify male donor cells in female recipients, and expression of the panendothelial marker mouse endothelial cell antigen-32 to identify sinusoidal endothelium; and (c) use of Tie-2/green fluorescent protein mice as donors or recipients and presence of Dil-Ac-LDL to identify sinusoids. We found that sinusoids were predominantly host-derived posttransplant. Donor cells spread along the marrow vasculature early post-transplant in a pattern that matched stromal-derived factor-1 expression. Furthermore, these engrafting progenitors were positioned to provide physical support, as well as growth and survival signals in the form of vascular-endothelial growth factor-A. Occasionally, donor cells provide cellular "patches" in the damaged sinusoids, although this occurred at a low level compared with hematopoietic engraftment. Donor support for the repair of the marrow vascular niche may be a critical first step of hematopoietic engraftment.
Objective MafG is the small subunit of the transcription factor NF-E2 that controls terminal megakaryocyte maturation and platelet release. Studies were conducted to evaluate the intrinsic and ...extrinsic effects of mafG deficiency on bone marrow engraftment kinetics. Materials and Methods We used mafG knockout mice either as donors or recipients in bone marrow transplantations with wild-type mice and compared the engraftment kinetics to transplantations using wild-type donors and recipients. We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight on day 5, 7, 14, and 1 month post-transplantation. Results Compared to wild-type recipients, mafG recipients had delayed platelet and leukocyte recovery and lower spleen weight at early time points after transplantation. Intrinsic effects: When mafG-deficient bone marrow served as donor source, we observed more rapid recovery of bone marrow cellularity and increased splenic hematopoiesis. The finding of increased short-term hematopoietic stem cells and progenitors in the mafG-deficient bone marrow could explain the accelerated hematopoietic recovery after transplantation. Furthermore, the expression of Bach 2, which can form a heterodimer with mafG protein, was found to be greatly reduced, while Notch 1 expression was increased in mafG-deficient mice. Extrinsic effects: When mafG-deficient mice were transplant recipients, there were delays in recovery of normal levels of marrow and splenic hematopoiesis as well as circulating leukocytes and platelets. Conclusions Our study demonstrates that mafG expression has intrinsic and extrinsic effects on hematopoietic engraftment following bone marrow transplantation.
Neuromuscular disorders such as Pompe disease (glycogen storage disease, type II), result in early and potentially irreversible cellular damage with a very limited opportunity for intervention in the ...newborn period. Pompe disease is due to deficiency in acid α-glucosidase (GAA) leading to lysosomal accumulation of glycogen in all cell types, abnormal myofibrillogenesis, respiratory insufficiency, neurological deficits, and reduced contractile function in striated muscle. Previous studies have shown that fetal delivery of recombinant adeno-associated virus (rAAV) encoding GAA to the peritoneal cavity of Gaa
mice resulted in high-level transduction of the diaphragm. While progression of other genetic disorders may occur later in life, the potential of fetal gene delivery to avoid the onset of irreversible damage suggests it is an attractive option for many inherited diseases. In this study, rhesus monkey fetuses were administered 4.5 × 10
particles of rAAV type 1 expressing human GAA (rAAV1-CMV-hGAA), human α-1-antitrypsin (rAAV1-CBA-hAAT), or human mini-dystrophin (rAAV1-CMV-miniDMD) in the late first trimester using an established intraperitoneal ultrasound-guided approach. Fetuses were monitored sonographically and newborns delivered at term for postnatal studies. All animals remained healthy during the study period (growth, hematology, and clinical chemistry), with no evidence of adverse effects. Tissues were collected at a postnatal age of 3 months (∼7 months post-fetal gene transfer) for immunohistochemistry (IHC) and quantitative PCR. Both the diaphragm and peritoneum from vector-treated animals were strongly positive for expression of human GAA, AAT, or dystrophin by IHC, similar to findings when reporter genes were used. Protein expression in the diaphragm and peritoneum correlated with high vector copy numbers detected by real-time PCR. Other anatomical areas were negative, although the liver showed minimal evidence of human GAA, AAT, and DMD, vector genomes. In summary, delivery of rAAV vectors provided stable transduction of the muscular component of the diaphragm without any evidence of adverse effects.
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