Primitive Precursor Cells Research Articles

Primitive and Definitive Neural Precursor Cells Are Present in Human Cerebral Organoids.

Activation of neural stem cells (NSCs) correlates with improved functional outcomes in mouse models of injury. In the murine brain, NSCs have been extensively characterized and comprise (1) primitive NSCs (pNSCs) and (2) definitive NSCs (dNSCs). pNSCs are the earliest cells in the NSC lineage giving rise to dNSCs in the embryonic and adult mouse brain. pNSCs are quiescent under baseline conditions and can be activated upon injury. Herein, we asked whether human pNSCs and dNSCs can be isolated during the maturation of human cerebral organoids (COs) and activated by drugs known to regulate mouse NSC behavior. We demonstrate that self-renewing, multipotent pNSC and dNSC populations are present in human COs and express genes previously characterized in mouse NSCs. The drug NWL283, an inhibitor of apoptosis, reduced cell death in COs but did not improve NSC survival. Metformin, a drug used to treat type II diabetes that is known to promote NSC activation in mice, was found to expand human NSC pools. Together, these findings are the first to identify and characterize human pNSCs, advancing our understanding of the human NSC lineage and highlighting drugs that enhance their activity.

Distinct immune escape and microenvironment between RG-like and pri-OPC-like glioma revealed by single-cell RNA-seq analysis

The association of neurogenesis and gliogenesis with glioma remains unclear. By conducting single-cell RNA-seq analyses on 26 gliomas, we reported their classification into primitive oligodendrocyte precursor cell (pri-OPC)-like and radial glia (RG)-like tumors and validated it in a public cohort and TCGA glioma. The RG-like tumors exhibited wild-type isocitrate dehydrogenase and tended to carry EGFR mutations, and the pri-OPC-like ones were prone to carrying TP53 mutations. Tumor subclones only in pri-OPC-like tumors showed substantially down-regulated MHC-I genes, suggesting their distinct immune evasion programs. Furthermore, the two subgroups appeared to extensively modulate glioma-infiltrating lymphocytes in distinct manners. Some specific genes not expressed in normal immune cells were found in glioma-infiltrating lymphocytes. For example, glial/glioma stem cell markers OLIG1/PTPRZ1 and B cell-specific receptors IGLC2/IGKC were expressed in pri-OPC-like and RG-like glioma-infiltrating lymphocytes, respectively. Their expression was positively correlated with those of immune checkpoint genes (e.g., LGALS33) and poor survivals as validated by the increased expression of LGALS3 upon IGKC overexpression in Jurkat cells. This finding indicated a potential inhibitory role in tumor-infiltrating lymphocytes and could provide a new way of cancer immune evasion.

Acute Alcohol Intoxication Impairs Sonic Hedgehog-Gli1 Signaling and Activation of Primitive Hematopoietic Precursor Cells in the Early Stage of Host Response to Bacteremia.

Activation of hematopoietic stem cells [HSCs, lineage(lin)- stem cell growth factor receptor (c-kit)+ stem cell antigen-1(Sca-1)+ , or LKS cells in mice] is critical for initiating the granulopoietic response. This study determined the effect of alcohol exposure on sonic hedgehog (SHH) signaling in the regulation of HSC activation during bacteremia. Acute alcohol intoxication was induced in mice by intraperitoneal (i.p.) injection of 20% alcohol (5g alcohol/kg body weight). Control mice received i.p. saline. Thirty minutes later, mice were intravenously (i.v.) injected with Escherichia coli (E.coli, 1 to 5×107 CFUs/mouse) or saline. SHH expression by lineage-negative bone marrow cells (BMCs) was significantly increased 24hours after E.coli infection. Extracellular signal-regulated kinase 1/2 (ERK1/2)-specificity protein 1 (Sp1) signaling promotes SHH expression. ERK1/2 was markedly activated in BMCs 8hours following E.coli infection. Alcohol suppressed both the activation of ERK1/2 and up-regulation of SHH expression following E.coli infection. E.coli infection up-regulated GLI family zinc finger 1 (Gli1) gene expression by BMCs and increased Gli1 protein content in LKS cells. The extent of Gli1 expression was correlated with the activity of proliferation in LKS cells. Alcohol inhibited up-regulation of Gli1 expression and activation of LKS cells in response to E.coli infection. Alcohol also interrupted the granulopoietic response to bacteremia. These data show that alcohol disrupts SHH-Gli1 signaling and HSC activation in the early stage of the granulopoietic response, which may serve as an important mechanism underlying the impairment of immune defense against bacterial infection in host excessively consuming alcohol.

KCa3.1 Channels Promote Cardiac Fibrosis Through Mediating Inflammation and Differentiation of Monocytes Into Myofibroblasts in Angiotensin II -Treated Rats.

BackgroundCardiac fibrosis is a core pathological process associated with heart failure. The recruitment and differentiation of primitive fibroblast precursor cells of bone marrow origin play a critical role in pathological interstitial cardiac fibrosis. The KC a3.1 channels are expressed in both ventricular fibroblasts and circulating mononuclear cells in rats and are upregulated by angiotensin II. We hypothesized that KC a3.1 channels mediate the inflammatory microenvironment in the heart, promoting the infiltrated bone marrow–derived circulating mononuclear cells to differentiate into myofibroblasts, leading to myocardial fibrosis.Methods and ResultsWe established a cardiac fibrosis model in rats by infusing angiotensin II to evaluate the impact of the specific KC a3.1 channel blocker TRAM‐34 on cardiac fibrosis. At the same time, mouse CD4+ T cells and rat circulating mononuclear cells were separated to investigate the underlying mechanism of the TRAM‐34 anti–cardiac fibrosis effect. TRAM‐34 significantly attenuated cardiac fibrosis and the inflammatory reaction and reduced the number of fibroblast precursor cells and myofibroblasts. Inhibition of KC a3.1 channels suppressed angiotensin II–stimulated expression and secretion of interleukin‐4 and interleukin‐13 in CD4+ T cells and interleukin‐4– or interleukin‐13–induced differentiation of monocytes into fibrocytes.Conclusions KC a3.1 channels facilitate myocardial inflammation and the differentiation of bone marrow‐derived monocytes into myofibroblasts in cardiac fibrosis caused by angiotensin II infusion.

Sonic Hedgehog Signaling Regulates Hematopoietic Stem/Progenitor Cell Activation during the Granulopoietic Response to Systemic Bacterial Infection.

Activation and reprogramming of hematopoietic stem/progenitor cells play a critical role in the granulopoietic response to bacterial infection. Our current study determined the significance of Sonic hedgehog (SHH) signaling in the regulation of hematopoietic precursor cell activity during the host defense response to systemic bacterial infection. Bacteremia was induced in male Balb/c mice via intravenous injection (i.v.) of Escherichia coli (5 × 107 CFUs/mouse). Control mice received i.v. saline. SHH protein level in bone marrow cell (BMC) lysates was markedly increased at both 24 and 48 h of bacteremia. By contrast, the amount of soluble SHH ligand in marrow elutes was significantly reduced. These contrasting alterations suggested that SHH ligand release from BMCs was reduced and/or binding of soluble SHH ligand to BMCs was enhanced. At both 12 and 24 h of bacteremia, SHH mRNA expression by BMCs was significantly upregulated. This upregulation of SHH mRNA expression was followed by a marked increase in SHH protein expression in BMCs. Activation of the ERK1/2–SP1 pathway was involved in mediating the upregulation of SHH gene expression. The major cell type showing the enhancement of SHH expression in the bone marrow was lineage positive cells. Gli1 positioned downstream of the SHH receptor activation serves as a key component of the hedgehog (HH) pathway. Primitive hematopoietic precursor cells exhibited the highest level of baseline Gli1 expression, suggesting that they were active cells responding to SHH ligand stimulation. Along with the increased expression of SHH in the bone marrow, expression of Gli1 by marrow cells was significantly upregulated at both mRNA and protein levels following bacteremia. This enhancement of Gli1 expression was correlated with activation of hematopoietic stem/progenitor cell proliferation. Mice with Gli1 gene deletion showed attenuation in activation of marrow hematopoietic stem/progenitor cell proliferation and inhibition of increase in blood granulocytes following bacteremia. Our results indicate that SHH signaling is critically important in the regulation of hematopoietic stem/progenitor cell activation and reprogramming during the granulopoietic response to serious bacterial infection.

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Probing for a deeper understanding of rhabdomyosarcoma: insights from complementary model systems.

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy composed of neoplastic primitive precursor cells that exhibit histological features of myogenic differentiation. Despite intensive conventional multimodal therapy, patients with high-risk RMS typically suffer from aggressive disease. The lack of directed therapies against RMS emphasizes the need to further uncover the molecular underpinnings of the disease. In this Review, we discuss the notable advances in the model systems now available to probe for new RMS-targetable pathogenetic mechanisms, and the possibilities for enhanced RMS therapeutics and improved clinical outcomes.

Neural Ganglioside GD2+ Cells Define a Subpopulation of Mesenchymal Stem Cells in Adult Murine Bone Marrow

Background/Aims: Due to the lack of specific markers, the isolation of pure mesenchymal stem cells (MSCs) from murine bone marrow remains an unsolved problem. The present study explored whether the neural ganglioside GD2 could serve as a single surface marker to uniquely distinguish murine bone marrow MSCs (mBM-MSCs) from other marrow elements. Methods: Immunocytochemistry and flow cytometry, in combination with quantitative RT-PCR, were used to identify the expression of GD2 on culture-expanded mBM-MSCs. GD2⁺ and GD2^- fractions from mBM-MSCs cultures were sorted by immunosorting. Flow cytometry was performed to further analyze the biomarkers of GD2-sorted and unsorted cells. Employing CFU-F assay and CCK-8 assay, we examined the clonogenic and proliferative capabilities of GD2-sorted and unsorted cells. Using oil red O and von Kossa staining assay, we also assessed the multi-lineage potential of GD2-sortedand unsorted cells. Results: We found that mBM-MSCs expressed a novel surface marker the neural ganglioside GD2. Importantly, mBM-MSCs were the only cells within bone marrow that expressed this marker. Further studies demonstrated that a homogenous population of MSCs could be obtained from bone marrow cultures in early passages by GD2 immunosorting. Compared to parental cells, GD2⁺-sorted cells not only possessed much higher clonogenic and proliferative capabilities but also had significantly stronger differentiation potential to adipocytes and osteoblasts. Furthermore, GD2⁺-sorted cells displayed enhanced expression of ES markers SSEA-1 and Nanog. Conclusion: Our observations provide the first demonstration that GD2 may serve as a maker for identification and purification of mBM-MSCs. Meanwhile, our study indicates that the cells selected by GD2 are a subpopulation of MSCs with features of primitive precursor cells.

Alcohol Administration Suppresses LKS Cell Proliferation During the Granulopoietic Response to E. Coli Bacteremia

Introduction: Excessive alcohol consumption injures the immune system, predisposing the host to the development of severe infections. Indeed, alcoholic patients with systemic bacterial infection frequently present with granulocytopenia which is an indicator for increased mortality. How alcohol impairs the granulopoietic response remains unclear. Methods: To determine whether alcohol affects the increase in primitive hematopoietic precursor cell commitment to granulocyte lineage development in response to bacteremia, acute alcohol intoxication was induced in male Balb/c mice maintained on a Lieber-DeCarli low fat liquid alcohol diet for 5 weeks by intraperitoneal injection (i.p.) of alcohol (20% alcohol in saline at a dose of 5 g alcohol/kg).

Mafb Restricts M-CSF Instructed Lineage Choice in HSC

AbstractAbstract SCI-15HSC need to enter the cycle to continuously regenerate mature blood cells in a correctly balanced ratio or to replenish the stem cell pool under stress conditions. Cell division of HSC may thus result in self-renewal divisions or the production of more differentiated progeny. Although such downstream progenitors still retain a high degree of multi-potency, recent advances in their characterization also suggest that early diversification into cells with distinct lineage bias can occur at the most primitive stem and precursor cell level. Whereas multiple cellular regulators have been identified that affect self-renewal of HSC, regulators that selectively control lineage specific commitment divisions of HSC are only beginning to be elucidated. Both transcription factor and cytokine signaling may play important roles in lineage engagement but it has been a long-standing debate, whether cytokine signaling has instructive or permissive effects on lineage commitment. In this context we reported that deficiency for the monocytic transcription factor MafB specifically sensitized HSC populations to M-CSF induced cell division and myeloid lineage commitment. We observed that MafB deficiency resulted in a specific up-regulation of the early myeloid transcription factor PU.1 and a dramatically enhanced myeloid specific repopulation activity that did not affect self-renewal or differentiation into other lineages. Our results point to a role for MafB in the maintenance of a balanced lineage potential of HSC by selectively restricting myeloid commitment divisions that give rise to PU.1+ progenitors in response to M-CSF signaling. Together this suggests that the potential of stem cells to produce differentiated progeny of a specific lineage bias can be subject to control by integrated cytokine/transcription factor circuits, where variation in cell intrinsic sensitivity limits like those set by MafB can render external cues such as M-CSF instructive.Stem cell self renewal or differentiation is associated with two different types of cell divisions, namely symmetric divisions that generate two identical daughter cells, or asymmetric divisions that produce a daughter cell with maintained stem cell properties and a daughter cell committed to a specific differentiation pathway. Our observation that reduced MafB levels specifically increased M-CSF stimulated asymmetric divisions giving rise to PU.1−/PU.1+ daughter pairs provides a conclusive explanation why increased myeloid commitment of MafB−/− HSC does not come at the expense of self-renewal or other lineages. The micro-environment can play a critical role in specifying the symmetry of cell divisions, but so far, investigations have focused on niche micro-environments that support HSC self-renewal. It is unknown whether environments also exist that support HSC commitment to specific lineages by enabling asymmetric divisions at the niche border. Using video-imaging approaches and genetic manipulation of the stem cell and stromal cell compartments we explore how cues from the microenvironment can affect asymmetric HSC division and lineage commitment. Disclosures:No relevant conflicts of interest to declare.

Embryonic Stem Cells Proliferate and Differentiate when Seeded into Kidney Scaffolds

The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Our goal was to decellularize intact organs in a manner that retained the matrix signal for differentiating pluripotent cells. We decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures. Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation. Cells not in contact with the basement membrane matrix became apoptotic, thereby forming lumens. These observations suggest that the extracellular matrix can direct regeneration of the kidney, and studies using seeded scaffolds may help define differentiation pathways.

Neural Ganglioside GD2 Identifies a Subpopulation of Mesenchymal Stem Cells in Umbilical Cord

In contrast to hematopoietic stem cells, there is still a lack of definitive cell markers for specific isolation and identification of mesenchymal stem cells (MSCs). Thus a homogenous population of MSCs is only obtained after several passages, when multilineage potential or other distinctive features of very early progenitors may be already somewhat compromised. Recently a novel surface marker the neural ganglioside GD2 has been reported to distinguish MSCs from all other cells within marrow. Here, we found that MSCs derived from umbilical cord (UC-MSCs) also expressed this marker at early-passages. More importantly, UC-MSCs were the only cells within umbilical cord expressing this marker. Compared to unsorted cells, GD2⁺-sorted cells not only possessed much higher clonogenicity and proliferation capacity but also had significantly stronger multi-differentiation potentials. Flow cytometric analysis revealed that GD2⁺-sorted cells showed increased expression of SSEA-4, Oct-4, Sox-2 and Nanog, the typical markers expressed in embryonic stem cells, in comparison to unsorted or GD2-negative MSCs. Take together, our data demonstrate that the cells selected by GD2 are a subpopulation of MSCs with feature of primitive precursor cells and provide evidence that GD2 can be a cell surface marker suitable for the isolation and purification of UC-MSCs in early-passage culture.

Lhx2 Expression Promotes Self-Renewal of a Distinct Multipotential Hematopoietic Progenitor Cell in Embryonic Stem Cell-Derived Embryoid Bodies

The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs) in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2−/− mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES) cell-derived embryoid bodies (EBs) can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

Common critical pathways in embryogenesis and cancer

Cancer may arise because the developmental programs that create the dramatic alterations in form and structure in embryonic development are potentially corrupted. The cells in our bodies retain memories of these processes and cancer can occur later in life if imperfections occur in the fidelity of these pathways. This article is particularly interested in the phenomenon of epithelial to mesenchymal transition, which occurs in embryogenesis. Also reviewed are the small molecules and pathways that are involved both in homeostasis in adult epithelium and embryogenesis in utero. There are five such pathways in particular selected for review in this article: the Wnt pathway, Hedgehog, Notch, PAR and Bone morphogenetic peptide/TGF β. These are usually conserved throughout mammalian evolution. Though they have been arbitrarily separated in this article they are not exclusive from one another. Their pathologically altered expression is found especially frequently in childhood tumours where they may recapitulate their developmental role, and in tumours that resemble primitive precursor cells. These pathways are important for selecting cell fates, cellular rearrangements, cytological context and morphologic design in embryology as well as participating in epithelial function in adults.

Proteolytic Activation of the Chemokine CCL15 by Cathepsin G from Neutrophil Granulocytes Modulates Proliferation of Primitive Hematopoietic Precursor Cells.

The human chemokine HCC-2 (MIP-1δ, small inducible cytokine subfamily A [Cys-Cys], member 15, hmrp-2b, MIP-5, NCC-3) is a potent chemoattractant for monocytes, lymphocytes and endothelial cells, and has been shown to inhibit proliferation of primitive hematopoietic precursor cells (HPC). CCL15 is constitutively expressed in gut and liver, and was identified to circulate in human plasma. The length of the N-terminus of CCL15 was found to control biological activity. We therefore aimed to investigate the processing of CCL15 in the hematopoietic system, and the effect of processed isoforms on HPC proliferation. Incubation of full-length CCL15 with cell-free bone marrow supernatant prepared from normal untreated mice revealed a decrease in molecular weight by approximately 3 kD. In contrast, the molecular weight of CCL15 remained unchanged when cell-free bone marrow supernatant of mice with regenerating hematopoiesis after 5 Gy gamma irradiation was analyzed. Incubation of full-length CCL15 with isolated neutrophils, but not monocytes, lymphocytes or thrombocytes revealed N-terminal deletions of 21 (Δ21), 23 (Δ23) and 26 (Δ26) amino acids, as assessed by Edman sequencing/mass spectrometry. Both (1) inhibition of the neutrophil-derived CCL15 proteolytic activity by protease inhibitors Suc-Ala-Ala-Pro-Phe-pNA, secretory leucoprotease inhibitor or elastatinal, and (2) addition of the recombinant serine proteases cathepsin G or elastase to full-length CCL15 revealed that the Δ23 and Δ26 isoforms are produced by cathepsin G, whereas formation of the Δ21isoform is dependent on elastase. In addition, the Δ23 and Δ26 isoforms of CCL15 was also directly isolated and sequenced from blood filtrate (hemofiltrate) collected from renal insufficiency patients. The Δ26 isoform was synthesized and analyzed in HPC proliferation assays. Compared to full length CCL15, the Δ26 isoform displayed a mean 2.5 fold increased potency to inhibit CFU-A colony formation from post 5-fluorouracil murine bone marrow. In addition, the Δ26 isoform efficiently abrogated cytosine arabinoside-induced suicide of murine spleen colony forming unit (CFU-S) in post 5-fluorouracil murine bone marrow, and inhibited growth of human cord blood CD34+ single cell clones induced by hematopoietic growth factors SCF, IL-3, GM-CSF and EPO after flow-cytometric cell sorting (mean 2.3 fold inhibition). Our findings indicate a role for neutrophil-derived cathepsin G in growth control of colony of primitive hematopoietic progenitor cells through the proteolytic processing of CCL15.

Chronic myeloid leukaemia: stem cell derived but progenitor cell driven

The biology of CML (chronic myeloid leukaemia) has been extensively investigated as the disease is a paradigm of neoplasms induced when a translocation results in expression of a novel fusion protein, in this instance p210(BCR-ABL). Although CML manifests itself principally as unregulated expansion of the myeloid lineage, the lesion is present in the stem cell population and it has long been assumed that disregulated stem cell kinetics must underlie the basic pathology of the disease. In this review, we present evidence that, in normal haemopoiesis, less primitive precursor cells retain considerable flexibility in their capacity to undergo self-renewal, allowing them to maintain lineage-specific homoeostasis without inflicting proliferative stress upon the stem cell population. This mechanism is dysregulated in CML and we have developed a self-renewal assay for CFU-GM (colony-forming unit-granulocyte/macrophage) which demonstrates that, in CML, the PI (proliferative index) of the myeloid progenitor cell population is increased. The ability to measure the PI as an endpoint of p210(BCR-ABL) expression gives considerable versatility to the in vitro investigation of putative therapeutic regimes in CML.

SDF-1 tells stem cells to mind their P’s and Ζ’s

Stromal cell-derived factor-1 (SDF-1) is a chemokine with unique functions, including a role in the trafficking of primitive blood precursor cells. A better understanding at a molecular level of how the binding of SDF-1 to its cell surface receptor, CXCR4, elicits specific biological responses in these cells has now been achieved through the identification of PKC-zeta activation as a common downstream signal. This finding suggests that treatment of a variety of clinical conditions might benefit from the targeting of PKC-zeta.

Effect of parathyroid hormone PTH (1-34) on hemopoietic and stromal stem cells

Long-term administration of parathyroid hormone causing activation and proliferation of osteoblasts to mice increases the concentration of primitive hemopoietic precursor cells (cobblestone area-forming cells) in long-living bone marrow culture after 28-35 days. The concentrations of later precursors forming colonies in the spleen and the concentration of cobblestone-area forming cells in long-living bone marrow culture after 7 days decrease, while the concentration of more differentiated cells forming colonies in the culture does not change. Transplantation of the bone marrow from mice treated with parathyroid hormone under the renal capsule of syngeneic recipients results in the formation of a focus of ectopic hemopoiesis not differing by size from the control. Injection of parathyroid hormone to mice during the growth of the ectopic focus did not modulate its size. These foci tolerate retransplantation procedure similarly as controls. Hence, parathyroid hormone has no effect on mesenchymal stem cells responsible for transfer of the stromal microenvironment. Therefore, the number of stem hemopoietic cells in the body is regulated by not stromal stem cells, but their better differentiated descendants.

Molecular characterization of conditionally immortalized cell lines derived from mouse early embryonic inner ear

Inner ear sensory hair cells (HCs), supporting cells (SCs), and sensory neurons (SNs) are hypothesized to develop from common progenitors in the early embryonic otocyst. Because little is known about the molecular signals that control this lineage specification, we derived a model system of early otic development: conditionally immortalized otocyst (IMO) cell lines from the embryonic day 9.5 Immortomouse. This age is the earliest stage at which the otocyst can easily be separated from surrounding mesenchymal, nervous system, and epithelial cells. At 9.5 days post coitum, there are still pluripotent cells in the otocyst, allowing for the eventual identification of both SN and HC precursors--and possibly an elusive inner ear stem cell. Cell lines derived from primitive precursor cells can also be used as blank canvases for transfections of genes that can affect lineage decisions as the cells differentiate. It is important, therefore, to characterize the "baseline state" of these cell lines in as much detail as possible. We characterized seven representative "precursor-like" IMO cell populations and the uncloned IMO cells, before cell sorting, at the molecular level by polymerase chain reaction (PCR) and immunocytochemistry (IHC), and one line (IMO-2B1) in detail by real-time quantitative PCR and IHC. Many of the phenotypic markers characteristic of differentiated HCs or SCs were detected in IMO-2B1 proliferating cells, as well as during differentiation for up to 30 days in culture. These IMO cell lines represent a unique model system for studying early stages of inner ear development and determining the consequences of affecting key molecular events in their differentiation.

Cellular approaches to tissue repair in cardiovascular disease: the more we know, the more there is to learn.

the rapidly advancing field of cellular plasticity bears exciting implications for both basic understanding of cell biology and potentially important therapeutic approaches to a range of disease states that remain as areas of great patient need. Indeed, the use of stem cells, progenitor cells, or