scholarly journals Genetic analysis reveals cell type-specific regulation of receptor tyrosine kinase c-Kit by the protein tyrosine phosphatase SHP1.

1996 ◽  
Vol 184 (3) ◽  
pp. 1111-1126 ◽  
Author(s):  
U Lorenz ◽  
A D Bergemann ◽  
H N Steinberg ◽  
J G Flanagan ◽  
X Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Genetic Analysis ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Receptor Protein ◽  
Cell Type ◽  
Protein Tyrosine ◽  
Cell Type Specific

Receptor protein tyrosine kinases (RTKs) transmit downstream signals via interactions with secondary signaling molecules containing SH2 domains. Although many SH2-phosphotyrosyl interactions have been defined in vitro, little is known about the physiological significance of specific RTK/SH2 interactions in vivo. Also, little is known about the mechanisms by which specific RTKs interact with and/or are regulated by specific protein tyrosine phosphatases (PTPs). To address such issue, we carried out a genetic analysis of the previously reported biochemical interaction between the RTK c-Kit, encoded at the W locus, and the SH2-containing non-transmembrane PTP SHP1, encoded at the motheaten (me) locus (1). Mice carrying a kinase-defective allele of c-Kit (Wv/+) were crossed with me/+ mice, which carry one effectively null allele of SHP1, and then backcrossed to generate all possible allelic combinations. Our results indicate strong intergenic complementation between these loci in hematopoietic progenitor cells. Compared to progenitors purified from normal mice, bone marrow progenitor cells (lin-) from me/me mice markedly hyper-proliferated in response to Kit ligand (KL). stimulation. Superimposition of the me/me genotype increased the number of one marrow-derived CFU-E from Wv/+ mice. Conversely, the presence of one or two copies of Wv decreased the number of macrophages and granulocytes in me/me lung, skin, peripheral blood and bone marrow, thereby decreasing the severity of the me/me phenotype. The decrease in dermal mast cells in Wv/Wv mice was rescued to levels found in Wv/+mice by superimposition of the me/me genotype. Surprisingly, however, the presence or absence of SHP1 had no effect on the proliferative response of bone marrow-derived cultured mast cells to KL or IL3 ex vivo. Nevertheless, the immediate-early response to KL stimulation, as measured by KL-induced tyrosyl phosphorylation, was substantially increased in mast cells from Wv/+:me/me compared to Wv/ +:+/+ mice, strongly suggesting that SHP1 directly dephosphorylates and regulates c-Kit. Taken together, our results establish that SHP1 negatively regulates signaling from c-Kit in vivo, but in a cell type-specific manner.

scholarly journals Functional disruption of α4 integrin mobilizes bone marrow–derived endothelial progenitors and augments ischemic neovascularization

2006 ◽  
Vol 203 (1) ◽  
pp. 153-163 ◽  
Author(s):  
Gangjian Qin ◽  
Masaaki Ii ◽  
Marcy Silver ◽  
Andrea Wecker ◽  
Evelyn Bord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
Cell Surface Receptor ◽  
Ischemic Disease ◽  
Angiogenic Therapy ◽  
Ischemic Tissue ◽  
Surface Receptor

The cell surface receptor α4 integrin plays a critical role in the homing, engraftment, and maintenance of hematopoietic progenitor cells (HPCs) in the bone marrow (BM). Down-regulation or functional blockade of α4 integrin or its ligand vascular cell adhesion molecule-1 mobilizes long-term HPCs. We investigated the role of α4 integrin in the mobilization and homing of BM endothelial progenitor cells (EPCs). EPCs with endothelial colony-forming activity in the BM are exclusively α4 integrin–expressing cells. In vivo, a single dose of anti–α4 integrin antibody resulted in increased circulating EPC counts for 3 d. In hindlimb ischemia and myocardial infarction, systemically administered anti–α4 integrin antibody increased recruitment and incorporation of BM EPCs in newly formed vasculature and improved functional blood flow recovery and tissue preservation. Interestingly, BM EPCs that had been preblocked with anti–α4 integrin ex vivo or collected from α4 integrin–deficient mice incorporated as well as control cells into the neovasculature in ischemic sites, suggesting that α4 integrin may be dispensable or play a redundant role in EPC homing to ischemic tissue. These data indicate that functional disruption of α4 integrin may represent a potential angiogenic therapy for ischemic disease by increasing the available circulating supply of EPCs.

Negative regulation of activated α2 integrins during thrombopoiesis

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6428-6439 ◽  
Author(s):  
Zhiying Zou ◽  
Alec A. Schmaier ◽  
Lan Cheng ◽  
Patricia Mericko ◽  
S. Kent Dickeson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Surface Expression ◽  
Wild Type ◽  
Vessel Injury ◽  
Rapid Signaling ◽  
Α2 Receptors

Abstract Circulating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, α2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface α2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, α2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated α2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated α2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoietic cells during migration between the adhesive environment of the bone marrow and the nonadhesive environment of the circulating blood.

scholarly journals In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Blood ◽  
2005 ◽  
Vol 106 (8) ◽  
pp. 2655-2662 ◽  
Author(s):  
Bianling Liu ◽  
Judy Daviau ◽  
Carmen N. Nichols ◽  
David S. Strayer
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Simian Virus 40 ◽  
Hematopoietic Stem ◽  
Entire Study

AbstractHematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)–derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope—or a control recombinant SV40 (rSV40)—directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study—56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.

scholarly journals Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice

2022 ◽  
pp. 074873042110694
Author(s):  
Ciearra B. Smith ◽  
Vincent van der Vinne ◽  
Eleanor McCartney ◽  
Adam C. Stowie ◽  
Tanya L. Leise ◽  
...  
Keyword(s):  
Locomotor Activity ◽  
Circadian Rhythms ◽  
Ex Vivo ◽  
Luciferase Expression ◽  
Dependent Manner ◽  
Cell Type ◽  
Reporter Mice ◽  
Mouse Tissues ◽  
Cell Type Specific

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein ( Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;Dbp KI/+ “liver reporter” mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

scholarly journals Cell type-specific isolation and transcriptomic profiling informs glial pathology in human temporal lobe epilepsy

2020 ◽  
Author(s):  
Jessica Tome-Garcia ◽  
German Nudelman ◽  
Zarmeen Mussa ◽  
Elodia Caballero ◽  
Yan Jiang ◽  
...  
Keyword(s):  
Temporal Lobe Epilepsy ◽  
Temporal Lobe ◽  
Ex Vivo ◽  
Hybrid Population ◽  
Rna Seq ◽  
Cell Type ◽  
Fresh Frozen ◽  
Cell Type Specific

The pathophysiology of epilepsy underlies complex network dysfunction, the cell-type-specific contributions of which remain poorly defined in human disease. In this study, we developed a strategy that simultaneously isolates neuronal, astrocyte and oligodendroglial progenitor (OPC)-enriched nuclei from human fresh-frozen neocortex and applied it to characterize the distinct transcriptome of each cell type in temporal lobe epilepsy (TLE) surgical samples. Differential RNA-seq analysis revealed several dysregulated pathways in neurons, OPCs, and astrocytes, and disclosed an immature phenotype switch in TLE astrocytes. An independent single cell RNA-seq TLE dataset uncovered a hybrid population of cells aberrantly co-expressing canonical astrocyte and OPC-like progenitor markers (GFAP+OLIG2+ glia), which we corroborated in-situ in human TLE samples, and further demonstrated their emergence after chronic seizure injury in a mouse model of status epilepticus. In line with their immature signature, a subset of human TLE glia were also abnormally proliferative, both in-vivo and in-vitro. Generally, this analysis validates the utility of the proposed cell type-specific isolation strategy to study glia-specific changes ex vivo using fresh-frozen human samples, and specifically, it delineates an aberrant glial phenotype in human TLE specimens.

Abstract 134: Low Dose Particle Radiation Affects Long-Term Survival of Bone Marrow Progenitor Cell Populations

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Sharath Sasi ◽  
Daniel Park ◽  
Maria A Zuriaga ◽  
Kenneth Walsh ◽  
Xinhua Yan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Particle Radiation ◽  
Term Survival ◽  
Time Points

Radiation-induced decreases in the number of bone marrow (BM)-derived endothelial progenitor cell (BM-EPCs) and their lineage precursors which include Early- and Late-Multi-Potent Progenitor cells (E-MPP and L-MPP) could contribute to the pathogenesis of ischemic and vascular diseases. We examined the effect of full-body single dose of proton (1H) at 0.5 Gy, 1 GeV and 0.15 Gy, 1 GeV/nucleon of iron (56Fe) - ionizing radiation (IR) on survival and proliferation of BM-EPCs. The survival of E-MPPs and L-MPPs in the BM after particle IR in C57BL/6 mice were determined at 1, 2, 4, 8, 12, 28 and 40 weeks post-IR. BM-derived mononuclear cells were triple-stained with RAM34 (CD34, c-kit, and Sca1), AC133, and hematopoietic lineage negative cocktail, then sorted by FASC for E- and L-MPP. BM EPCs ex-vivo - There was a transient 2.5-3.5-fold increase in BM-EPC apoptosis, with 3.5-fold increases for 56Fe and 1H at 5hrs and 24hrs, respectively that was no longer detected by day 7. Subsequently, there was a 3-fold increase in BM-EPC apoptosis on day 28 for both ion-IR mice. Compared to 24 hrs, there was a ~20% (1H) and ~45% (56Fe) increase in the rate of EPC proliferation on day 14 that returned to control levels on day 28. BM E-MPP and L-MPP in vivo - Compared to control mice, 1H-IR increased the number of both E-MPPs (665%) and L-MPPs (203%), whereas 56Fe-IR decreased E-MPP (74%) and L-MPPs (65%) at 1 week post-IR, suggesting stimulation by 1H but overt damage by 56Fe in the BM milieu. In 56Fe-IR mice, E-MPPs recovered between 4 and 12 weeks, followed by declines at later time points. In 1H-IR mice, E-MPPs were near control levels up to 4 weeks, but declined at later time points. The long-lasting and cyclical effects of IR on the BM E- and L-MPPs after a single 1H or 56Fe IR dose suggests the presence of prolonged and non-targeted effects in BM milieu, that occur in cells that were not traversed by IR, rather induced by signals from IR cells. Our studies showed that, both 1H- and 56Fe-IR has profound and long-lasting (28-40 months) negative effects on the number of E- and L-MPPs. Future longitudinal studies are necessary to determine whether BM progenitor cells may be affected after terrestrial IR exposure, such as cancer radiotherapy, CT and PET scans, and in astronauts after exploration-type space missions.

ITag-RNA Allows in Vivo Cell-Type Specific Transcriptional Characterization and Tracking of Circulating Transcripts

2018 ◽  
Author(s):  
J. Darr ◽  
M. Lassi ◽  
R. Gerlini ◽  
F. Scheid ◽  
M. Hrabě de Angelis ◽  
...  
Keyword(s):  
Cell Type ◽  
Cell Type Specific

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

Differential pial and penetrating arterial responses examined by optogenetic activation of astrocytes and neurons

2021 ◽  
pp. 0271678X2110103
Author(s):  
Nao Hatakeyama ◽  
Miyuki Unekawa ◽  
Juri Murata ◽  
Yutaka Tomita ◽  
Norihiro Suzuki ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Acetylcholine Receptors ◽  
Step Function ◽  
Muscarinic Acetylcholine Receptors ◽  
Cell Type ◽  
Function Type ◽  
Neuron Activation ◽  
Cell Type Specific ◽  
Arterial Responses

A variety of brain cells participates in neurovascular coupling by transmitting and modulating vasoactive signals. The present study aimed to probe cell type-dependent cerebrovascular (i.e., pial and penetrating arterial) responses with optogenetics in the cortex of anesthetized mice. Two lines of the transgenic mice expressing a step function type of light-gated cation channel (channelrhodopsine-2; ChR2) in either cortical neurons (muscarinic acetylcholine receptors) or astrocytes (Mlc1-positive) were used in the experiments. Photo-activation of ChR2-expressing astrocytes resulted in a widespread increase in cerebral blood flow (CBF), extending to the nonstimulated periphery. In contrast, photo-activation of ChR2-expressing neurons led to a relatively localized increase in CBF. The differences in the spatial extent of the CBF responses are potentially explained by differences in the involvement of the vascular compartments. In vivo imaging of the cerebrovascular responses revealed that ChR2-expressing astrocyte activation led to the dilation of both pial and penetrating arteries, whereas ChR2-expressing neuron activation predominantly caused dilation of the penetrating arterioles. Pharmacological studies showed that cell type-specific signaling mechanisms participate in the optogenetically induced cerebrovascular responses. In conclusion, pial and penetrating arterial vasodilation were differentially evoked by ChR2-expressing astrocytes and neurons.

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