David Trisler

Hematopoietic progenitors express neural genes

Bone marrow, or cells selected from bone marrow, were reported recently to give rise to cells with a neural phenotype after in vitro treatment with neural-inducing factors or after delivery into the brain. However, we showed previously that untreated bone marrow cells express products of the neural myelin basic protein gene, and we demonstrate here that a subset of ex vivo bone marrow cells expresses the neurogenic transcription factor Pax-6 as well as neuronal genes encoding neurofilament H, NeuN (neuronal nuclear protein), HuC/HuD (Hu-antigen C/Hu-antigen D), and GAD65 (glutamic acid decarboxylase 65), as well as the oligodendroglial gene encoding CNPase (2′,3′ cyclic nucleotide 3′-phosphohydrolase). In contrast, astroglial glial fibrillary acidic protein (GFAP) was not detected. These cells also were CD34+, a marker of hematopoietic stem cells. Cultures of these highly proliferative CD34+ cells, derived from adult mouse bone marrow, uniformly displayed a phenotype comparable with that of hematopoietic progenitor cells (CD45+, CD34+, Sca-1+, AA4.1+, cKit+, GATA-2+, and LMO-2+). The neuronal and oligodendroglial genes expressed in ex vivo bone marrow also were expressed in all cultured CD34+ cells, and GFAP was not observed. After CD34+ cell transplantation into adult brain, neuronal or oligodendroglial markers segregated into distinct nonoverlapping cell populations, whereas astroglial GFAP appeared, in the absence of other neural markers, in a separate set of implanted cells. Thus, neuronal and oligodendroglial gene products are present in a subset of bone marrow cells, and the expression of these genes can be regulated in brain. The fact that these CD34+ cells also express transcription factors (Rex-1 and Oct-4) that are found in early development elicits the hypothesis that they may be pluripotent embryonic-like stem cells.

Brain derived neurotrophic factor treatment reduces inflammation and apoptosis in experimental allergic encephalomyelitis

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) which includes a neurodegenerative component. Brain derived neurotrophic factor (BDNF) is a neuroprotective agent which might be useful in preventing neurodegeneration but its application has been limited because the blood brain barrier restricts its access to the CNS. We have developed a novel delivery system for BDNF using transformed bone marrow stem cells (BMSC) and undertook studies of EAE to determine whether the delivery of BDNF could reduce inflammation and apoptosis. Mice receiving BDNF producing BMSC had reduced clinical impairment compared to control mice receiving BMSC that did not produce BDNF. Pathological examination of brain and spinal cord showed a reduction in inflammatory infiltrating cells in treated compared to control mice. Apoptosis was reduced in brain and spinal cord based on TUNEL and cleaved Caspase-3 staining. Consistent with the known mechanism of action of BDNF on apoptosis, Bcl-2 and Akt were increased in treated mice. Further studies suggested that these increases could be mediated by inhibition of both caspase dependent and caspase independent pathways. These results suggest that the BDNF delivered by the transformed bone marrow stem cells reduced clinical severity, inflammation and apoptosis in this model.

Brain-derived neurotrophic factor gene delivery in an animal model of multiple sclerosis using bone marrow stem cells as a vehicle

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is neuroprotective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the central nervous system (CNS), when delivered peripherally, is limited due to the blood-brain barrier (BBB). We have developed a means of delivering BDNF into the CNS using genetically engineered bone marrow stem cells (BMSCs) as a vehicle, and have explored the clinical effects of BDNF on outcomes in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). BDNF-engineered-BMSCs were transplanted (i.v.) into irradiated 2-week-old SJL/J female mice. Eight weeks after transplantation, mice were immunized with a peptide of proteolipid protein (PLP(139-151)). Mice, which had received BDNFengineered BMSCs, showed a significant delay in EAE onset and a reduction in overall clinical severity compared to mice receiving BMSC transfected with an empty vector lacking the BDNF gene. In addition, pathological examination showed that BDNF delivery reduced demyelination and increased remyelination. Inhibition of pro-inflammatory cytokines TNF-alpha and IFN-gamma and enhanced expression of the antiinflammatory cytokines IL-4, IL-10, and IL-11 were found in the CNS tissues of the BDNF transplanted group. These results support the use of BMSCs as vehicles to deliver BDNF into the CNS of EAE animals. This is a potentially novel therapeutic approach that might be used to deliver BDNF gene or genes for other therapeutic proteins into the CNS in MS or in other diseases of the CNS in which accessibility of therapeutic proteins is limited due to the BBB.

Overexpression of SIRT1 Protein in Neurons Protects against Experimental Autoimmune Encephalomyelitis through Activation of Multiple SIRT1 Targets

Treatment of experimental autoimmune encephalomyelitis (EAE) with resveratrol, an activator of sirtuin 1 (SIRT1), reduces disease severity. This suggested that activators of SIRT1, a highly conserved NAD-dependent protein deacetylase, might have immune-modulating or neuroprotective therapeutic effects in EAE. Previously, we showed that SIRT1 expression increases in EAE, suggesting that it is an adaptive response. In this study, we investigated the potential function of SIRT1 in regulating EAE using SIRT1-overexpressing mice. The current studies examine potential neuroprotective and immunomodulatory effects of SIRT1 overexpression in chronic EAE induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein peptide 35–55. SIRT1 suppressed EAE clinical symptoms compared with wild-type EAE mice and prevented or altered the phenotype of inflammation in spinal cords; as a result, demyelination and axonal injury were reduced. Significant neuroprotective effects were observed, with fewer apoptotic cells found in the spinal cords of SIRT1-overexpressing EAE mice associated with increased brain-derived neurotrophic factor and NAD levels. Earlier, we showed that brain-derived neurotrophic factor and NAD play crucial neuroprotective roles in EAE. These results suggest that SIRT1 reduces neuronal loss in this chronic demyelinating disease model and that this is associated with a reduction in inflammation.

Stem cell based delivery of IFN-β reduces relapses in experimental autoimmune encephalomyelitis ☆

Interferon-beta (IFN-beta), an approved treatment of multiple sclerosis (MS), produces only partial clinical responses. IFN-beta therapy has been limited by its short serum half-life and limited ability to cross the blood brain barrier. We have developed a means of delivering the IFN-beta gene both systemically and into the central nervous system (CNS) using bone marrow stem cells (BMSCs) as a vehicle and examined the therapeutic efficacy of this approach in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. A retroviral expression vector (pLXSN-IFNbeta) was used to stably transfect virus producer PA317 cells to generate retrovirus containing the IFN-beta gene which then was used to transduce BMSCs. IFN-beta engineered BMSCs were transplanted (i.v.) into mice that then were immunized with proteolipoprotein (PLP) to initiate EAE. IFN-beta-engineered BMSCs transplanted mice showed a significant inhibition of EAE onset, and the overall clinical severity was less compared to control groups. IFN-beta delivery strongly reduced infiltration of mononuclear cells possibly by inhibiting cell adhesion molecules. Reduced demyelination and increased remyelination were also observed in the IFN-beta treated group. Furthermore, inhibition of the pro-inflammatory cytokines TNF-alpha, IFN-gamma and IL-12 and enhanced expression of the anti-inflammatory cytokines IL-10, IL-4 and TGF-beta was observed in CNS tissue. In addition, mice receiving IFN-beta had reduced apoptosis and increases in growth promoting factors including BDNF, CNTF, PDGF and VEGF. These results suggest that BMSCs can be used as vehicles to deliver the IFN-beta into the CNS. This is a potentially novel therapeutic approach which might be used in MS and other diseases of the CNS in which drug access is limited.

Brain-derived neurotrophic factor (BDNF) gene delivery into the CNS using bone marrow cells as vehicles in mice

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is protective in animal models of neurodegenerative diseases. However, BDNF has a short half-life and its efficacy in the CNS when delivered peripherally is limited due to the blood-brain barrier. In the present study, bone marrow cells were used as vehicles to deliver the BDNF gene into the CNS. Marrow cells obtained from 6 to 8 week-old SJL/J mice were transduced with BDNF expressing pro-virus. RT-PCR analysis revealed that BDNF mRNA was expressed in transduced but not in non-transduced marrow cells. Additionally, virus transduced marrow cells expressed the BDNF protein (296+/-1.2 unit/ml). BDNF-transduced marrow cells were then transplanted into irradiated mice through the tail vein. Three months post-transplantation, significant increases in BDNF as well as glutamic acid decarboxylase (GAD(67)) mRNA were detected in the brains of BDNF transplanted mice compared to untransplanted animals, indicating biological activity of the BDNF transgene. Thus, bone marrow cells can be used as vehicles to deliver the BDNF gene into the brain with implications for the treatment of neurological diseases.

TrkB agonist, 7,8-dihydroxyflavone, reduces the clinical and pathological severity of a murine model of multiple sclerosis

7,8-Dihydroxyflavone (DHF), is a recently described TrkB agonist that readily crosses the blood brain barrier. We treated C57Bl/6 mice with MOG--induced EAE daily with DHF starting on the day of disease induction. Clinical severity of impairment was reduced throughout the course of disease. Pathological examination of brains and spinal cords on day 28 showed that DHF treatment increased the phosphorylation of TrkB and activated downstream signaling pathways including AKT and STAT3 and reduced inflammation, demyelination and axonal loss compared to EAE controls. DHF treatment duplicated the central nervous system effects of brain derived neurotrophic factor in the EAE.

Use of engineered bone marrow stem cells to deliver brain derived neurotrophic factor under the control of a tetracycline sensitive response element in experimental allergic encephalomyelitis

Brain derived neurotrophic factor (BDNF) has neuroprotective properties but its use has been limited by poor penetration of the blood brain barrier. Treatment using bone marrow stem cells (BMSC) or retroviruses as vectors reduces the clinical and pathological severity of experimental allergic encephalomyelitis (EAE). We have refined the BMSC based delivery system by introducing a tetracycline sensitive response element to control BDNF expression. We have now tested that construct in EAE and have shown a reduction in both the clinical and pathological severity of the disease. Further, we looked for changes in sirtuin1 and nicotinamide phosphoribosyltransferase expression that would be consistent with a neuroprotective effect.

Hematopoietic progenitors express embryonic stem cell and germ layer genes

Cell therapy for tissue regeneration requires cells with high self-renewal potential and with the capacity to differentiate into multiple differentiated cell lineages, like embryonic stem cells (ESCs) and adult somatic cells induced to pluripotency (iPSCs) by genetic manipulation. Here we report that normal adult mammalian bone marrow contains cells, with the cell surface antigen CD34, that naturally express genes characteristic of ESCs and required to generate iPSCs. In addition, these CD34+ cells spontaneously express, without genetic manipulation, genes characteristic of the three embryonic germ layers: ectoderm, mesoderm and endoderm. In addition to the neural lineage genes we previously reported in these CD34+ cells, we found that they express genes of the mesodermal cardiac muscle lineage and of the endodermal pancreatic lineage as well as intestinal lineage genes. Thus, these normal cells in the adult spontaneously exhibit characteristics of embryonic-like stem cells.

Adult hematopoietic progenitors are multipotent in chimeric mice.

Embryonic stem cells (ESCs) and adult somatic cells, induced to pluripotency (iPSCs), can differentiate into multiple cell lineages. We previously reported that adult mammalian bone marrow contains a sub-population of CD34+ cells that express genes of ESCs and genes required to generate iPSCs. They also express lineage genes of the three embryonic germ layers. Are these CD34+ cells multipotent? Here, CD34+ bone marrow stem cells from adult male ROSA mice, which carry two markers: the β-galactosidase gene and the male Y chromosome, were transplanted into blastocysts of wildtype mice. Each female ROSA chimera generated had a distinct pattern of male-derived organs expressing β-galactosidase; e.g., ectodermal brain, dorsal root ganglia and skin; mesodermal heart, bone and bone marrow; and endodermal pancreas, intestine, and liver. Thus, adult mammals carry cells that appear to exhibit a developmental potential reminiscent of ESCs and iPSCs suggesting they could be used for cell replacement therapy.