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There is interest in the use of Mesenchymal stem cells/marrow stromal cells (MSCs) to treat neurodegenerative trinucleotide repeat disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease (HD). MSCs present a promising tool for cell therapy, and are currently being tested in FDA-approved Phase I to III clinical trials for many disorders. They have been extensively tested and proven effective in pre-clinical studies for neurodegenerative disorders. Studies have examined the potential benefits from the innate trophic support from MSC or from augmented growth factor support, such as delivering brain-derived neurotrophic factor (BDNF) or glial-derived neurotrophic factor (GDNF) into the brain to support injured neurons, using genetically engineered MSC as the delivery vehicles. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals and regulate inflammation, primarily through paracrine actions. MSC transplanted into the brain have been shown to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. Clinical trials for MSC injection into the central nervous system to treat Amyotrophic Lateral Sclerosis (ALS), traumatic brain injury and stroke are currently ongoing. The current protocols are provided in support of applying MSC-based cellular therapies to the treatment of trinucleotide repeat disorders.
There is currently much interest in the use of MSC to treat neurodegenerative diseases, in particular those like Huntington's disease that are fatal and difficult to treat, through providing neurotrophic factors to encourage repair and potentially new growth of neurons from neural progenitor cells. Proposed regenerative approaches include delivery via intracerebral or intrathecal injection or even infusion via an intranasal route1. Therapies will capitalize upon the innate trophic support from MSC or on augmented growth factor support, such as delivering brain-derived neurotrophic factor (BDNF) or glial-derived neurotrophic factor (GDNF) into the brain to support injured neurons, using genetically engineered MSC as the delivery vehicles.
Upon transplantation, MSC in the brain promote endogenous neuronal growth, decrease apoptosis, and regulate inflammation, primarily through the use of secreted factors. MSC can mediate modification of the damaged tissue microenvironment to enhance endogenous neural regeneration and protection. MSC transplanted at sites of nerve injury have been shown to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons2. Transplantation of human bone marrow stem cells into the brain of immunodeficient mice markedly increased the proliferation of endogenous neural stem cells3. In an experimental allergic encephalomyelitis model of multiple sclerosis, rodents that received an intraventricular infusion of mesenchymal stem cells were found to have almost twice the number of axons as control animals.4 Although candidate molecules are under investigation, further detailed studies are needed to carefully define the factors responsible for the MSC-mediated induction of proliferation in resident neural stem cells, in order to best capitalize upon this type of therapy for the repair of neurodegenerative diseases.
An interesting development candidate on which we and others are working for consideration of treatment of the trinucleotide repeat disorder Huntington's disease (HD) is allogeneic human MSC engineered to secrete BDNF as a neurotrophic strategy to enhance neural survival and regeneration. BDNF levels are very low in mice and humans with HD. In rodent models, BDNF has been shown to ameliorate symptoms and to extend survival, and therefore BDNF therapy is a leading candidate for use in treating HD. Striatal neurons depend on BDNF levels for function and survival.5 In the later stages of HD, available BDNF levels plummet since the mutant protein prevents production at the RNA level.6, 7 This reduction of BDNF levels affects the onset and severity of the disease in HD mice.5 Up-regulation of BDNF in the brains of transgenic rodent models of HD has shown amelioration of the disease phenotype (reviewed by Zuccato et al8). Due to its pro-survival effects in striatal neuropathology, BDNF is a leading candidate for neuroprotective therapies in HD (reviewed by Alberch9).
In transgenic mouse models there is a significant reduction in behavioral phenotype following administration of MSC/BDNF.10 The Dunbar laboratory has shown that MSCs, especially those engineered to over-express BDNF, have significant ameliorative effects on disease progression in a transgenic mouse model of HD.10 These studies have demonstrated that MSCs engineered to produce BDNF and implanted into the striata significantly increased time to fall in rotorod testing, demonstrating an increased coordination and reduction in movement disorders in treated mice, over the 13 months of the study. The MSC/BDNF also significantly reduced limb clasping, a hallmark behavioral defect in HD mice, over the same time period.10
A major impediment for the clinical use of neurotrophic factors is their inability to cross the blood-brain barrier in therapeutic amounts. In the quest to begin translating promising studies such as those published by Dey et al10 to the clinic, we have shown biosafety of intracranial administration of MSCs in long term rodent and non-human primate models (MS in preparation, 2011). Our group uses human MSC as safe and long-lived delivery factories for cytokines and growth factors. We are currently evaluating the safety and efficacy of MSC engineered to secrete BDNF in peripheral tissues and in the brain to enhance neurorestorative capacity in HD.
The use of MSC to deliver factors, both through their own innate responses and through engineering, has benefits over direct protein administration, because transplanted MSCs can provide sustained and long-term delivery of factors at supraphysiological levels, as we and others have shown over the past two decades.11–17 Using immune deficient mouse models, we have recovered human MSC from numerous organs, including the brain, at timepoints from 1–18 months post-transplantation, with continued expression of the gene product.11, 12, 14, 16, 18–20. We also performed a decade-long biosafety study to demonstrate that genetically engineered human MSC are safe and do not cause adverse events in vivo.21 Efforts from our laboratory and others are currently evaluating the effects of BDNF expression from human MSC implanted into the striata of immunocompromised HD mice. Data from our lab and others provide support for the potential for MSCs to safely deliver augmented neurotrophic support in the central nervous system.
The potential benefit to risk ratio and in particular safety are always the foremost considerations by the Food and Drug Administration. This review has covered the potential benefits of MSC-based therapies for the treatment of neurodegenerative diseases. We and many others have documented biosafety of MSC therapies, which are now in phase III trials for some indications, and further safety and efficacy data are being collected by numerous groups. Extending MSC-based therapies to neurodegenerative diseases, in particular those for which there are currently no effective treatments, such as Huntington's disease, would have a high potential benefit to risk ratio. The need for safe and effective cellular therapies to treat Huntington's disease is great, since current therapies only target symptoms and there are currently no drugs or other treatments that effectively delay the relentless loss of striatal volume in affected patients.
350 ml Iscove's Modified Dulbecco's Medium (IMDM)
75 ml heat- inactivated (HI) horse serum*
75 ml HI Fetal calf serum*
5 ml L-glutamine (200 mM stock)
2.5 ml Pen/Strep (stock = 10,000 U/ml penicillin and 10,000 ug/ml streptomycin)
500 ul 2-ME (10−1 M stock)
500 ul hydrocortisone (10−3 M stock)
450 ml Dulbecco's Modified Eagles medium with high glucose
50 ml heat- inactivated (HI) Fetal calf serum*
5 ml L-glutamine (200 mM stock)
2.5 ml Pen/Strep (stock = 10,000 U/ml penicillin and 10,000 ug/ml streptomycin)
The screens used to filter marrow during harvest are the richest source of mesenchymal stem cells, as we have described 11, 18. Many small bony spicules packed with stroma (as well as hematopoietic stem and progenitor cells) will get lodged in the screen, and can be easily removed by flushing. The cells from one harvest screen, from a normal donor, should be split between 4 × T-75 vent-cap flasks in 15 mls of stromal medium (section 3) per flask. The cells will then be expanded, as described below (section 3). Filter vent-cap flasks are used for long-term culture, despite their greater cost as compared to standard screw-caps, because the risks for air-borne fungal spore contamination are high for cultures which can be grown for 1–2 months. The tightly closed, gas permeable filter vent caps reduce the risk of cross-contamination between flasks.
Many transplant programs are now using G-CSF mobilized peripheral blood as a stem cell source in lieu of bone marrow. Unfortunately, MSC are not found in appreciable levels in G-CSF mobilized blood. The use of newer mobilization agents, such as the CXCR4 antagonist AMD3100 (Mozobil) 22, might provide better MSC mobilization than the standard regimens. Until then, researchers at those institutions can purchase whole marrow, or even purified and cryopreserved MSC, from commercial sources, or might consider beginning a normal donor program for marrow donation to be used for research. In lieu of flushing harvest screens, when whole marrow is available from these sources, the investigator should proceed as directed below.
If the richest source of MSC- bone marrow harvest screens- is not available, whole aspirated bone marrow can be used as a source of mesenchymal stem cells. Spicules from unseparated BM will be present in the aspirate, and can be collected by gravity sedimentation. The liquid marrow is then removed to another tube for additional processing. It is advisable to perform at least one red cell lysis and wash before plating, if using this method. The washing technique is described. The spicules from a 10 ml aspirate should then be plated in T-75 vent-cap flasks in 15 mls of stromal medium (Dexter's original medium = DOM, section 2), which is the richest medium and rapidly forces contaminating hematopoietic cells into erythroid and monocytic differentiation. A simpler medium can also be used, as described in section 2. If the aspirate providing the spicules is larger, the number of flasks should be scaled up accordingly. If the BM sample must be ficolled for other studies, and the MSC investigator is salvaging spicules, use the techniques described in the section below.
The ficolled “buffy coat” is not a rich source of MSC, but is what many investigators have to work with. The MSC are far more rare in the aspirated marrow fraction than in spicules from the harvest screens. They are even rarer if the sample is first ficolled (approximately 1 × 106/ ml in the “buffy coat” or mononuclear fraction. If whole marrow aspirates are to be used, an optimal strategy is to use the mononuclear fraction, and also to recover the spicules from the bottom of the 50 ml ficoll tubes, since the small pieces of bone will fall through the density gradient.
For ficolling, first mix an equal volume of whole marrow and 1× phosphate buffered saline and then gently layer 25 mls over an equal volume of ficoll-paque in a 50 ml conical tube. Centrifuge the cells at 2000 rpm for 15 minutes. Once approximately 15 mls of the serum layer has been removed and discarded, buffy coat cells can be collected in another 10–15 mls, washed, and plated as described below. Then there are 5–10 mls of packed red blood cells and bony spicules left in the bottom of the tube. PBS should then be added up to a volume of 50 mls. Allow the tubes to settle upright for 3 minutes, without centrifugation. Remove 40 mls PBS and RBC, then repeat the washing step: add PBS, let the spicules settle out, and remove RBC/PBS down to the final 10 mls. At this point, the red blood cells are sufficiently diluted out to allow plating of the spicules. Add another 40 mls PBS, centrifuge, remove fluid down to the last ml, and plate as described below.
If total (RBC lysed) or ficolled marrow is used, to expand mesenchymal stem cells, cells are plated at a concentration of 5 × 106 mononuclear cells per ml in 75 cm2 flasks, in 15–20 mls total volume. Optimally, plate the cells in Dexter's original medium (DOM), which is prepared as shown in the Materials section.
Preparation of spicules for plating is described above, in section 2. Spicules obtained from one harvest screen should be divided between four T-75 flasks containing 15 mls of DOM or D10HG each. Spicules obtained from the RBC pellet resulting from 10–15 mls of ficolled marrow can be plated in one T-75 flask, in 15 mls of medium. Cells are then expanded and transduced as described below.
Following the initial seeding, described in section 3, the MSC are allowed to adhere to the flasks overnight. The next morning, non-adherent cells can be gently flushed from the flasks and replated in a second flask, in the same medium. The initial flask is refed fresh medium. DOM is the richest medium for MSC expansion without differentiation, and the horse serum rapidly forces contaminating hematopoietic cells into erythroid and monocytic differentiation, so that hematopoietic stem cells will not contaminate the stromal layer after 3 passages. A minimal medium, D10HG, which contains only fetal calf serum, can also be used, but hematopoietic stem cells will survive happily on the stromal layer in this medium. MSC have not yet been expanded efficiently without the use of fetal calf serum, and it is imperative to screen the serum for optimal MSC growth without differentiation, when using either medium.
MSC colonies begin to develop as the cells expand out of the marrow spicules (Figure 1). There are many other cells in the culture at this point. However, as the MSC grow and expand, the other cells differentiate out and /or can be removed. In the fetal calf/horse serum mixture (DOM), the developing erythroid cells become non-adherent and are easily flushed away as the MSC layer develops and is expanded. Alternately, a depletion step can be done at passage 2–3, to remove Glycophorin A+ cells. Early monocytes can also be removed by flushing, but mature macrophages are tightly adherent to the tissue culture flask and cannot be removed, even with trypsin. Therefore, the MSC can be taken to a new flask, while leaving the macrophages behind to be discarded.
Alternately, the cells can be collected using an EDTA-based cell dissociation buffer (rather than trypsin, which cleaves away many cell surface proteins), and then a FACS-based depletion can be done to remove CD45+ cells, including CD14+ monocyte/macrophages, from the developing MSC monolayer.
If not using FACS to fractionate MSC subpopulations based on cell surface proteins, it is best to use trypsinization (trypsin-EDTA), to dissociate sub-confluent monolayers of primary mesenchymal stem cells from the flask. For general maintenance and expansion, the cells should be “split” no more than 1:10 when they reach 70–80% confluency (Figure 2).
MSCs are not usually transduced or used for other studies until passage #3 or 4. At this point (Figure 2), most hematopoietic cells will have been eliminated, except for mature macrophages which typically will comprise less than 1% of the culture.
The cells should be used for transduction, experiments, or transplantation between passage 3–6 for optimal results. By passage ten, they can begin to differentiate and become senescent. Since the primary MSC cultures are not immortalized, they do have a finite lifespan, and by later passage, they begin to slow down in growth and to become larger and more differentiated.
For retroviral transduction, add supernatant from MoMuLV- based retroviral vectors with protamine sulfate (final concentration = 4 ug/ml) four times, over a 48 hour period. Protamine sulfate is a polycationic compound which neutralizes the negatively charged retroviral particles and cell surfaces. Add it only once every 24 hours, or it will be toxic. This should result in 20–40% of the flask being transduced, due to the rapid division of the MSC. The cells must be subconfluent when each aliquot of supernatant is added. Confluent cells are contact inhibited and will not divide to allow retroviral vector integration.
VSV-G pseudotyped lentiviral vector supernatant can be added once or twice at an MOI of 10–100, without the need for protamine sulfate. Select the cells according to the selectable marker included in the chosen vector (if using G418 to select for the neo gene, the best concentration is 0.75 mg/ml active drug), or use as partially - transduced monolayers. Transduced MSC are excellent vehicles from which to secrete proteins, as we have described11, 16–18, 28.
While the methodologies for transducing MSC are relatively simple, since in log phase the cells are rapidly dividing and incorporate vector very easily, in comparison to hematopoietic stem cells 29–31, several cautions do exist for transduction and for reliably assessing the success of the MSC transduction. Although lentiviral vectors can enter non-dividing cells, MSC monolayers should still be subconfluent prior to transduction, or the VSV-G envelope can cause cell fusion, resulting in multinucleate cells which appear overnight in the culture.
An important caution in interpretation of transduction is that MSC can and do “share” proteins with neighboring cells, through junction formation or other as-yet-unknown mechanisms. For this reason, we have described that MSC (marrow stromal cells) must be plated at subconfluency for selective agents such as G418 to work effectively11, 18. This is also reflected in the fact that fluorescent markers such as eGFP can be shared between cells. Transduced cells dropped into a confluent plate of non-transduced MSC can cause a green “halo” to be seen in neighboring cells, although it is not as bright in intensity as seen in the cell that is expressing the transgene. For this reason, caution should be exercised when interpreting immunofluorescence or FACS data from partially transduced MSC cultures. If stringent parameters are set for the highly expressing cells, those that contain transgene product assimilated using the “bystander effect” will not be included. The propensity for MSC to share proteins is, however, a factor in making them excellent vehicles for delivering enzyme products or other transgenes to cells in a deficient animal or in an injured tissue.
Author Contributions: Geralyn Annett: Conception and design, Manuscript writing, Final approval of manuscript
Gerhard Bauer: Manuscript writing, Final approval of manuscript
Jan A. Nolta: Conception and design, Assembly of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript