Jueren Lou

Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats

Bone marrow derived mesenchymal stem cells (MSC) have been shown to be progenitor cells for mesenchymal tissues. These cells may also provide a potential therapy for bone repair. Our previous studies showed that MSC engineered with the gene for bone morphogenetic protein 2 (BMP‐2), a growth factor for bone cells, were capable of differentiating into osteoblast lineage and inducing autologous bone formation in several animal models. Culturing individual MSC for autologous implantation, however, remains problematic. The number of human MSC with osteogenic potential decreases with age, and, in certain diseases, the patients marrow may be damaged or the healthy cells reduced in number. In this study, we used rats with a femoral segmental defect to investigate whether allogeneic BMP‐2 engineered MSC would facilitate bone healing. We show that BMP‐2 engineered allogeneic MSC can repair critical bone defects to the same degree as rats treated with BMP‐2 engineered autologous MSC, if the allogeneic group receives short‐term treatment with immunosuppressant FK506. We also show that allogeneic gene transferred MSC are directly involved in bone repair, in addition to acting as gene deliverers.

Migration of Mesenchymal Stem Cells Through Cerebrospinal Fluid into Injured Spinal Cord Tissue

Study Design. Experimental spinal cord injury using a rat model. Objective. To investigate the potential for survival and migration of transplanted mesenchymal stem cells through the subarachnoid space into injured thoracic spinal cord tissue following injection into the more caudal lumbar spine. Methods. A total of 70 adult Lewis rats were used with 64 having a partial and complete thoracic spinal cord injury (SCI) performed by the weight drop method at T9–T10 using the NYU Impactor. Six rats received only laminectomy for sham control. Mesenchymal stem cells (MSCs) were harvested from the femur of these rats and labeled by transduction of ANOVA virus containing green fluorescent protein (GFP) gene (Adv-GFP). At day 3, 5, and 7 after thoracic SCI, the rats received an injection into the subarachnoid space. The injections including: GFP-MSC, B-Gal-MSC, and PBS only. Injured rat spinal cords where harvested at day 7, 14, or 28, prepared for frozen sectioning, staining, and immunostaining. Results. Adv-GFP transduced MSCs demonstrated strong green fluorescence both in the nucleus and in the cell body. Green fluorescent cells proven to be genuine GFP-positive cells were observed on the surface of the injured spinal cord parenchyma. The rate of the GFP-positive cells gathered into the central lesion within 10 mm was significantly higher than sham control. Also, GFP-positive cells were observed in the deeper area of the perivascular spaces, and some of them had integrated into the parenchyma. Immunostaining against Nestin demonstrated that some GFP-positive cells differentiated into neural stem cells and mature neurons or glial cells. Conclusions. Transplanted MSCs injected into the subarachnoid space of the lumbar spine can migrate to injured thoracic spinal cord tissue. The ratio of MSCs observed at the injury site was significantly higher than in the intact spinal cord, and also infiltrated into the deeper spinal cord parenchyma by the perivascular spaces. Lastly, some MSCs differentiated into Nestin-positive, immature neurons or glial cells.

Flexor tendon healing in the rat: a histologic and gene expression study☆

PURPOSE To establish a rat flexor tendon laceration and repair model to investigate the molecular mechanisms of flexor tendon healing. METHODS Surgery was performed on rat flexor digitorum longus tendons from both hind feet. Repaired tendons were harvested at 0, 3, 7, 14, 21, 28, 42, 56, and 84 days after surgery. Histologic study (first 84 days) and gene expression study (first 28 days) of several collagens and matrix metalloproteinases (MMPs) were performed. RESULTS In the histologic study pre-existing collagen bundles were degraded between days 7 to 21. Newly formed collagen fibers crossed the repair site by day 28. Remodeling of the collagen fibers continued until day 84. Gene expression of type I collagen decreased initially and then returned gradually to the initial level by day 28, whereas expression levels of types III, V, and XII collagen were increased after surgery. The expression levels of MMP-9 and MMP-13 peaked between days 7 to 14, whereas MMP-2, MMP-3, and MMP-14 levels increased after surgery and maintained high levels until day 28. CONCLUSIONS The rat tendon laceration model represented the entire tendon healing process. The results of this study suggest that MMP-9 and MMP-13 participate only in collagen degradation, whereas MMP-2, MMP-3, and MMP-14 participate not only in collagen degradation but also in collagen remodeling.

Apoptosis as a mechanism of neuronal cell death following acute experimental spinal cord injury.

The complex biochemical interactions following acute spinal cord injury have undergone considerable investigation recently. Progress has been made in discovering both primary and secondary injury cascades that combine to produce the ultimate neurologic insult. Traditionally, neuronal and supporting cell death following spinal cord injury have focused on necrotic death pathways resulting passively from the actual mechanical tissue damage and inflammatory processes which follow. However, the occurrence of programmed apoptotic cell death which is an actively mediated cellular process may occur following acute spinal cord injury and, if present, may play a role in the ultimate neurologic insult. In this study, we document a chronologically-specific course of apoptotic cell death by the TUNEL assay technique following an acute experimental spinal cord injury in the rat model. In this manner, apoptotic cell death following acute spinal cord injury may play a pivotal role in the secondary injury cascade which produces the ultimate neurologic insult and may allow potential for mediating neuronal survival via anti-apoptotic factors such as the proto-oncogene Bcl-2.

Quantitative variation in vascular endothelial growth factor mRNA expression during early flexor tendon healing: an investigation in a canine model

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis, with direct mitogenic activity on cells of endothelial origin. We quantified the temporal accumulation of VEGF mRNA at the repair site of an in vivo canine intrasynovial flexor tendon repair and rehabilitation model by means of quantitative Northern blot analysis, in order to detail a molecular signal involved in the intrinsic angiogenic process that accompanies early flexor tendon healing. Significant accumulation of VEGF mRNA occurred at the flexor tendon repair site at 7 days post‐operatively, with peak levels seen at post‐operative days 7 and 10. Levels returned to baseline by day 14.

Effect of bone morphogenetic protein-12 gene transfer on mesenchymal progenitor cells

Recombinant adenovirus mediated human bone morphogenetic protein-12 gene transfer induced tendon and cartilage-like tissue formation in vivo. The recombinant adenovirus with the human bone morphogenetic protein-12 gene was constructed, and mature human bone morphogenetic protein-12 expression mediated by adenovirus gene transfer was detected by specific antibody. Unlike bone morphogenetic protein-2 gene transfer, bone morphogenetic protein-12 gene transferred mesenchymal progenitor cell line C3H 10T1/2 showed no change of alkaline phosphatase activity, which is the mark of cell differentiation into osteoblastic phenotype. Injection of bone morphogenetic protein-12 gene transferred C3H 10T1/2 cells into nude mice thigh muscles induced tendon and cartilage-like tissue formation. The results indicate bone morphogenetic protein-12 has different effects on mesenchymal progenitor cell differentiation, and it may influence the cell differentiation into a nonosteoblast lineage.

astrocyte Response and Transforming Growth Factor-β Localization in Acute Spinal Cord Injury

Study Design. An experimental histologic and immunohistological investigation of acute spinal cord injury was performed in a rat model. Objective. This study determined (1) the immediate cellular and molecular responses within the spinal cord that result from a clinically relevant compression injury, (2) the acute astrocytic response to injury using the astrocyte specific GFAP antibody, and (3) the temporal pattern of cellular and extracellular localization of transforming growth factor‐&bgr;1 (TGF‐&bgr;1) within the spinal cord injury zone immediately after injury. Summary of Background Data. Ultimate neurologic outcome from spinal cord injury results from both the primary mechanical trauma and a subsequent cascade of cellular and molecular events that are termed the secondary injury. Efforts aimed at improving neurologic outcome may depend on the manipulation of cellular and molecular mechanisms that are responsible for propagating this secondary injury cascade. Astrocytes and TGF‐&bgr; are two potentially key components of this secondary injury. Methods. Twenty‐one Sprague‐Dawley adult rats underwent open thoracic spinal cord injuries using the Allen weight‐drop technique. Spinal cord specimens were harvested at 0, 1, 2, 4, 8, 24, and 72 hours after injury for histologic and immunohistochemical evaluation. Harvesting of injured and surrounding uninjured cord was performed before sectioning in sagittal and transverse planes. These paraffin‐embedded sections were stained with polyclonal antibodies against glial fibrillary acidic protein (GFAP, an astrocytic cvtoskeleton marker) and TGF‐01. Results. A complex astrocytic response to the spinal cord injury was found within 24 hours of injury. Both the geographic and temporal patterns of astrocyte localization suggest a role in the regulation of spinal cord injury propagation. High concentrations of extracellular TGF‐&bgr; were seen immediately after injury within the hematoma at the zone of impact. Subsequently, intracellular TGF‐&bgr; was seen in astrocytic nuclei and cytoplasm, intramedullary and extramedullar capillary endothelial cells, and in motor neurons. Conclusions. The neurologic outcome in patients with SCI results in part from a secondary injury whose cellular and molecular mechanisms are poorly understood. This study suggests that both astrocytes and TGF‐&bgr; are involved in the regulation of spinal cord secondary injury. An improved understanding of their specific roles may result in novel treatments to improve the outcome from SCI. [Key words: acute spinal cord injury, astrocytes, transforming growth factor‐&bgr;, glial fibrillary acidic protein] Spine 1994;19:2321‐2330

Optimal treatment timing to attenuate neuronal apoptosis via Bcl-2 gene transfer in vitro and in vivo.

Although Bcl-2 gene transfer can rescue cells from neuronal apoptosis, the temporal relationship between treatment initiation time and effectiveness is unknown. The purpose of present study is to investigate the optimal treatment timing of Bcl-2 gene transfer in saving cells after neural insults. Bcl-2 gene transfer was mediated by recombinant adenovirus carrying human bcl-2 oncogene (Adv-Bcl-2). Adenovirus carrying beta-galactosidase gene (Adv-Bgal) served as a control. A serum withdrawal model of NSC-19 cell culture was used to induce apoptosis in vitro. At various time points before or after serum withdrawal, the motor neuron cells (NSC-19 cells) were infected with either Adv-Bcl-2 or Adv-Bgal. At 72 h after serum withdrawal, the number of apoptotic cells and DNA fragmentation were examined to evaluate the effect of Bcl-2 gene transfer. A weight-drop spinal cord injury model in rats was used as in vivo model. At various time points before or after experimental spinal injury, virus solution, including Adv-Bcl-2 or Adv-Bgal, was injected at the spinal cord in injured rats. The degree of cord injury was measured at 72 h after injury. TUNEL staining was performed to count cells that have undergone DNA damage in sections. Bcl-2 protein overexpression was confirmed by immunostaining both in vitro and in vivo model. In vitro, Adv-Bcl-2 infection produced a less prominent DNA laddering pattern. Adv-Bcl-2 infection between 24 h before and 4 h after serum withdrawal significantly reduced the apoptotic cell death. In vivo Adv-Bcl-2 injection immediately after injury effectively suppressed the injury lesion by blocking DNA fragmentation and irreversible cellular injury. Our data demonstrate that earlier initiation of Bcl-2 gene transfer can produce improved neural cell rescue following neural insults. These results stress important temporal considerations in future gene therapy strategies for spinal cord injury.

The role of Sp1 in BMP2-up-regulated Erk2 gene expression ☆

Extracellular signal-regulated kinase (Erk) is an important component in many cellular processes, including cell differentiation and proliferation. We previously showed that Erk is involved in BMP2-induced osteoblastic differentiation in mesenchymal progenitor cells and Erk protein level is up-regulated under BMP2 inducement. In this study, the molecular mechanism which mediates the regulation of Erk2 gene expression by BMP2 was investigated. Northern blot analysis showed that increased Erk2 protein level under BMP2 inducement comes from BMP2-up-regulated Erk2 mRNA expression. Transient transfection of C3H10T1/2 cells with a series of constructs of mouse Erk2 promoter demonstrated that a sequence residing between nucleotides -148 and -42 of Erk2 promoter is one of the BMP2-responsive elements. Electrophoresis mobility shift assays indicated that BMP2 treatment on C3H10T1/2 cells increases the binding of cell nuclear extracts to the -148/-42 fragment, and the BMP2-enhanced binding bands are Sp1 transcription factors. A series of competitive gel shift assays and the supershift assays by mapping oligos S1-S5 on -148/-42 identified that S1 and S5 contain Sp1 binding sites, which are located, respectively, in -147/-139 and -51/-46. Transfection studies showed that the addition of the Sp1 binding inhibitor mithramycin or mutation of the Sp1 site residing at -147/-139 abolishes the up-regulation of Erk2 promoter activity induced by BMP2. All these results indicate that Sp1-mediated transcription is one of the mechanisms, which is responsible for BMP2-induced up-regulation of Erk2 expression.