Green Fluorescent Protein Chimeric Mice Research Articles

Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n=10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone induction in the future.

Distribution of Bone Marrow-Derived Cells in the Fracture Callus during Plate Fixation in a Green Fluorescent Protein-Chimeric Mouse Model

To clarify the distribution of bone-marrow-derived cells in fractures treated by plate fixation, fracture models were created using the green fluorescent protein (GFP) chimeric mouse. We observed 2 types of fracture healing processes with different types of callus formation and cellular events by using Mouse Fix™, a device allowing plate fixation on the mouse femur, and differences in the distribution of bone-marrow-derived cells between the 2 types. The GFP chimeric mice were created by bone marrow transplantation. Fractures were created on the left femurs of mice and stabilized with either rigid (Group R) or flexible (Group F) plates to prepare undecalcified fresh-frozen sections. In Group F, a large external callus and a large intramedullary callus were formed mostly by endochondral ossification. The cells that made up the intramedullary callus and callus in the fracture gap were GFP positive, but most cells of the external callus were not. In Group R, bone union was achieved mostly without external callus formation, bone apposition occurred directly in the gap, and a small intramedullary callus was formed. As observed in Group F, this group had GFP-positive cells in the callus within the fracture gap and in the intramedullary calluses. The results of this study provided direct evidence of the distribution of bone-marrow-derived cells in the callus of fractures treated by plate fixation under different stability conditions.

Corneal myofibroblast generation from bone marrow-derived cells

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP−, SMA−GFP+ and SMA−GFP− cells, as well as the number of DAPI+ cell nuclei, per 400× field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9× more SMA+GFP+ than SMA+GFP− myofibroblasts. This difference was significant ( p < 0.01). There were significantly more ( p < 0.01) SMA−GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP− cells, although SMA−GFP− cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60–95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.

Controlled Release of Bone Morphogenetic Protein-2 Enhances Recruitment of Osteogenic Progenitor Cells for De Novo Generation of Bone Tissue

The objective of this study was to evaluate the cellular contribution to the phenomenon of de novo generation of bone tissue induced by the controlled release of bone morphogenetic protein-2 (BMP-2). Gelatin hydrogels (2 mg) incorporating BMP-2 (3 microg) with different water contents were subcutaneously implanted into the back of enhanced green fluorescent protein-chimeric mice to induce the ectopic de novo generation of bone tissue. The hydrogels incorporating BMP-2 could release BMP-2 at different time profiles. When evaluated radiologically and histologically, the ectopic de novo generation of bone tissue was induced by the controlled release of BMP-2 from the hydrogels around the hydrogel-implanted site. The relative percentage number of green fluorescent protein- to osteocalcin-positive cells recruited into the de novo generated bone tissue depended on the BMP-2 release profile. The higher the percentage, the stronger was the de novo generation of bone tissue. These findings indicate that bone marrow-derived osteoblast progenitor cells were recruited from the blood circulation by BMP-2 release and consequently contributed to the ectopic de novo generation of bone tissue. It is conceivable that the local concentration of BMP-2 modifies the recruitment profile of progenitor cells with an osteogenic potential around the release site of BMP-2, resulting in regulated volume of de novo generated bone tissue.

Insulin-Like Growth Factor Binding Protein-3 Mediates Vascular Repair by Enhancing Nitric Oxide Generation

Insulin-like growth factor binding protein (IGFBP)-3 modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury. To examine the mechanism of IGFBP-3-mediated repair following vascular injury. We used 2 complementary vascular injury models: laser occlusion of retinal vessels in adult green fluorescent protein (GFP) chimeric mice and oxygen-induced retinopathy in mouse pups. Intravitreal injection of IGFBP-3-expressing plasmid into lasered GFP chimeric mice stimulated homing of EPCs, whereas reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous NO generation. Thus, we asked whether the migratory effects of IGFBP-3 were attributable to stimulation of NO generation. IGFBP-3 increased endothelial NO synthase expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator-stimulated phosphoprotein, an NO regulated protein critical for cell migration. IGFBP-3-mediated NO generation required high-density lipoprotein receptor activation and stimulation of phosphatidylinositol 3-kinase/Akt pathway. These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.

Stromal Cell-Derived Factor-1α Promotes Neuroprotection, Angiogenesis, and Mobilization/Homing of Bone Marrow-Derived Cells in Stroke Rats

Stromal cell-derived factor (SDF)-1alpha is involved in the trafficking of hematopoietic stem cells from bone marrow to peripheral blood, and its expression is increased in the penumbra of the ischemic brain. In this study, SDF-1alpha was found to exert neuroprotective effects that rescued primary cortical cultures from H(2)O(2) neurotoxicity, and to modulate neurotrophic factor expression. Rats receiving intracerebral administration of SDF-1alpha showed less cerebral infarction due to up-regulation of antiapoptotic proteins, and they had improved motor performance. SDF-1alpha injection enhanced the targeting of bone marrow (BM)-derived cells to the injured brain, as demonstrated in green fluorescent protein-chimeric mice with cerebral ischemia. In addition, increased vascular density in the ischemic cortex of SDF-1alpha-treated rats enhanced functional local cerebral blood flow. In summary, intracerebral administration of SDF-1alpha resulted in neuroprotection against neurotoxic insult, and it induced increased BM-derived cell targeting to the ischemic brain, thereby reducing the volume of cerebral infarction and improving neural plasticity.

Granulocyte colony-stimulating factor improves left ventricular function of doxorubicin-induced cardiomyopathy

It is not well-known yet how granulocyte colony-stimulating factor (G-CSF) affects nonischemic cardiomyopathy, though its beneficial effects on acute myocardial infarction are well-established. We hypothesize that G-CSF beneficially might affect nonischemic cardiomyopathy through the direct cardioprotective effects. Here, we show that a single injection of doxorubicin (DOX, 15 mg/kg) induced left ventricular dilatation and dysfunction in mice within 2 weeks, and that these effects were significantly attenuated by human recombinant G-CSF (100 μg/kg/day for 5 days). G-CSF also protected hearts against DOX-induced cardiomyocyte atrophy/degeneration, fibrosis, inflammatory cell infiltration and down regulation of GATA-4 and sarcomeric proteins, myosin heavy chain, troponin I and desmin, both in vivo and in vitro. Cardiac cyclooxygenase-2 was upregulated and G-CSF receptor was downregulated in DOX-induced cardiomyopathy, but both of those effects were largely reversed by G-CSF. No DOX-induced apoptotic effects were seen, nor were there any changes in tumor necrosis factor-α or transforming growth factor-β1 levels. Among downstream mediators of G-CSF receptor signaling, DOX-induced cardiomyopathy involved inactivation of extracellular signal-regulated protein kinase (ERK); the ERK inactivation was reversed by G-CSF. Inhibition of ERK activation, but not cyclooxygenase-2 inhibition, completely abolished beneficial effect of G-CSF on cardiac function. G-CSF did not promote differentiation of bone marrow cells into cardiomyocytes according to the experiment using green fluorescent protein-chimeric mice, and inhibition of CXCR4+ cell homing using AMD3100 did not diminish the effect of G-CSF. Finally, G-CSF was also effective when administered after cardiomyopathy was established. In conclusion, these findings imply the therapeutic usefulness of G-CSF mainly through restoring ERK activation against DOX-induced nonischemic cardiomyopathy.

Selective Ablation of Proliferating Microglial Cells Exacerbates Ischemic Injury in the Brain

Here we report in vivo evidence of a neuroprotective role of proliferating microglial cells in cerebral ischemia. Using transgenic mice expressing a mutant thymidine kinase form of herpes simplex virus driven by myeloid-specific CD11b promoter and ganciclovir treatment as a tool, we selectively ablated proliferating (Mac-2 positive) microglia after transient middle cerebral artery occlusion. The series of experiments using green fluorescent protein-chimeric mice demonstrated that within the first 72 h after ischemic injury, the Mac-2 marker [unlike Iba1 (ionized calcium-binding adapter molecule 1)] was preferentially expressed by the resident microglia. Selective ablation of proliferating resident microglia was associated with a marked alteration in the temporal dynamics of proinflammatory cytokine expression, a significant increase in the size of infarction associated with a 2.7-fold increase in the number of apoptotic cells, predominantly neurons, and a 1.8-fold decrease in the levels of IGF-1. A double-immunofluorescence analysis revealed a approximately 100% colocalization between IGF-1 positive cells and Mac-2, a marker of activated/proliferating resident microglia. Conversely, stimulation of microglial proliferation after cerebral ischemia by M-CSF (macrophage colony stimulating factor) resulted in a 1.9-fold increase in IGF-1 levels and a significant increase of Mac2+ cells. Our findings suggest that a postischemic proliferation of the resident microglial cells may serve as an important modulator of a brain inflammatory response. More importantly, our results revealed a marked neuroprotective potential of proliferating microglia serving as an endogenous pool of neurotrophic molecules such as IGF-1, which may open new therapeutic avenues in the treatment of stroke and other neurological disorders.

Treatment with granulocyte colony-stimulating factor ameliorates chronic heart failure

Chronic heart failure remains a leading cause of mortality. Although granulocyte colony-stimulating factor (G-CSF) is reported to have a beneficial affect on postinfarction cardiac remodeling and dysfunction when administered before the onset of or at the acute stage of myocardial infarction (MI), its effect on established heart failure is unknown. We show here that subcutaneous administration of G-CSF greatly improves the function of murine hearts failing due to a large, healed MI. G-CSF changed the geometry of the infarct scar from elongated and thin to short and thick, induced hypertrophy among surviving cardiomyocytes, and reduced myocardial fibrosis. Expression of G-CSF receptor was confirmed in failing hearts and was upregulated by G-CSF treatment. G-CSF treatment also led to activation of signal transducer and activator of transcription-3 and induction of GATA-4 and various sarcomeric proteins such as myosin heavy chain, troponin I and desmin. Expression of metalloproteinase-2 and -9 was also increased in G-CSF-treated hearts, while that of tumor necrosis factor-α, angiotensin II type 1 receptor (AT1) and transforming growth factor-β1 was reduced. Although activation of Akt was noted in G-CSF-treated hearts, vessel density was unchanged, and apoptosis was too rare to exert a meaningful effect. No bone marrow-derived cardiomyocytes or vascular cells were detected in the failing hearts of green fluorescent protein chimeric mice. Finally, beneficial effects of G-CSF on cardiac function were found persisting long after discontinuing the treatment (2 weeks). Collectively, these findings suggest G-CSF administration could be an effective approach to treating chronic heart failure following a large MI.

Cardiac Progenitor Cells

See related article, pages 1090–1092 In the last few years we have witnessed one of most extraordinary revolutions in cardiovascular medicine, namely, an explosion of basic and clinical studies that support the notion that the diseased heart can be repaired by administration of stem cells, resulting in formation of functional new myocytes and vessels. Although the mechanism by which cell therapy improves cardiac function and anatomy remains uncertain, translation of basic findings to the clinical setting is proceeding at a feverish pace.1 A multitude of small, mostly nonrandomized clinical studies have reported improvement in cardiac perfusion and function after therapy with various cell types in patients with acute myocardial infarction or chronic ischemic cardiomyopathy.1 Larger, randomized, double-blinded studies will be reported soon; if they confirm the salubrious effects of cell-based therapies observed in the initial trials, our management of acute myocardial infarction and heart failure will change dramatically. One of the most important and unresolved issues in this scenario is the identity of the ideal cell for myocardial reconstitution. Although most of the clinical studies reported to date have used bone marrow– or skeletal muscle–derived cells, a host of other cells are being investigated in the experimental laboratory. Among these, resident cardiac stem cells (CSCs), discovered by Anversa’s group in 2003,2 hold great promise. In their initial report,2 Beltrami et al identified a lin−/c-kit+ population of primitive cells that can be clonally expanded, differentiates into cardiac myocytes, smooth muscle cells, and endothelial cells in vitro, and is able to reconstitute infarcted myocardium in vivo. It is of translational interest that these cells can effect cardiac repair when delivered via the intravascular route.3 CSCs have also been shown to be present in the human heart, where they give rise to new myocytes in patients with …

The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses.

Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells.

Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas. Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation. In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas. Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed.