The present invention relates to novel methods to improve the survival of mesenchymal stem cells in vivo. These methods can be used to improve procedures and implants used in a variety of diseases where promotion of tissue repair is necessary for recovery or cure from disease.
Mesenchymal stem cells (MSCs) possess a broad spectrum of regenerative properties, which are being deployed in clinical trials to treat numerous disorders. MSC applications range from repairing articular cartilage defects to improving neurological function after a stroke. The success of MSC therapies is dependent upon the survival of implanted stem cells. Engraftment applications rely on MSCs to integrate and replace damaged or diseased tissue, while non-engraftment applications leverage the continued presence of MSCs to secrete bioactive factors that promote tissue repair. Accordingly, standardization of MSC survival in vivo is essential to achieve consistent treatment outcomes.
Transplanted MSCs are stressed in vivo by a variety of factors, including ischemia at the implant site. In this harsh environment, MSC implants survive in vivo for only days to weeks, whereas, repair of tissues like bone takes months. To illustrate, the amount of rat MSCs in allografts decreased over 70% after 3 days in vivo according to one study but experienced only a 50% loss after 35 days according to another report. As a result, MSC implants are viable for only a fraction of the healing time.
Previous attempts to improve MSC survival in vivo include preconditioning the stem cells prior to implantation with growth factors and hypoxia. Other strategies to retain viable MSC implants have focused on manipulating the concentration and attachment of bioactive molecules in the stem cell microenvironment.
The literature is silent on the contribution of cellular heterogeneity to the survival of MSC implants. MSC cultures are a heterogeneous mixture of progenitors with different regenerative potentials at different stages of cellular aging. Long-term culture of MSCs revealed continuous and incremental changes to their global gene expression profile towards a senescent phenotype, as cellular aging is a result of accumulated DNA damage from replicative stress and can result in a functional change that is detrimental to the regenerative properties of MSCs, including a decrease in proliferation potential. Although stem cell aging is being studied extensively in vitro, to date, there has been no work to investigate the in vivo survival of aging MSCs of any kind. This is a critical knowledge gap in light of the importance of cell survival to MSC therapies and the impaired proliferation potential of aging MSCs.
TRAIL receptor CD264 has been reported as the first known surface marker of cellular aging for MSCs. CD264 is upregulated concomitantly with p21 at an intermediate stage of cellular aging and remains upregulated through senescence. It is reported that MSC cultures from young donors contained 20-40% CD264+ cells, with even higher CD264+ cell content possible for older donor cultures. In addition, a strong inverse correlation of CD264+ cell content in MSC cultures with their in vitro proliferation and differentiation potential has also been reported.
On the other hand, while it has been reported that the level of NG2 expression positively correlates with the prolifieration and trilineage potential of MSCs in vitro, such correlation was never extended to in vivo MSC survival. as discussed above the in vivo conditions are different from in vitro conditions, and the stress response of cells to unfavorable environment activates different pathways to cope with potentially lethal stimuli.
Therefore, there is still the need for better screening method for in vivo MSC survival in order to improve the efficacy of MSC therapies.
A method of improving mesenchymal stem cells (MSCs) in vivo survival is described herein. After collecting MSCs from a subject, the first step is to select and isolate the MSCs with high expression of neuron glial antigen 2 (NG2Hi), as these MSCs are found to have the longest in vivo survival rate. The NG2Hi MSCs are then expanded to necessary amount for MSC therapy. Once the number of MSCs is sufficient, depending on the different treatment methods, a scaffold may be provided onto which the MSCs can attach. The resulting scaffold with attached MSCs can then be implanted into a patient.
A composition of MSCs having high in vivo survival rate is also described herein. In the composition, at least 50% of MSCs have high NG2 expression. The composition can also comprise less than 30% of CD264+ MSCs or more than 40% of CD264+ MSCs. In embodiment, the composition comprises less than 15% CD264+ MSCs, or less than 10% CD264+ MSCs. In embodiments, the composition comprises more than 50% CD264+ MSCs.
In embodiments, the NG2Hi MSCs are selected and isolated by flow cytometry, in which only the cells in the top 30% expression level are selected. In one embodiment, only cells in the top 15% expression level are selected. In one embodiment, only cells in the top 10% expression level are selected.
In embodiments, during the expansion step, CD264+ cells can be removed, such that only less than 30% of the expanded MSCs are CD264+. In one embodiment, only less than 15% of the expanded MSCs are CD264+. In one embodiment, only less than 10% of the expanded MSCs are CD264+.
In embodiments, during the expansion step, CD264+ cells can be enriched, such that at least 40% of the expanded MSCs are CD264+. In one embodiment, at least 50% of the expanded MSCs are CD264+.
In embodiments, after the desired therapeutic effect has been reached, or after a predetermined period of time, the CD264+ MSCs are removed or killed from the scaffold.
A method of implanting mesenchymal stem cells in a mammal is described, comprising the steps of: a) selecting and/or isolating, from a heterogeneous group of MSCs, MSCs having top 30% expression level of neuron-glial antigen 2 (NG2); b) expanding the MSCs selected in step a), wherein the expanded MSCs having CD264+ are removed or killed; c) attaching the MSCs expanded in step b) to a scaffold; and d) implanting the scaffold with the MSCs in a mammal.
In embodiments, after the removal of CD264+ MSCs, only 15% or less of the expanded MSCs are CD264+. In embodiments, after the removal of CD264+ MSCs, only 10% or less of the expanded MSCs are CD264+.
A method of implanting mesenchymal stem cells in a mammal is described, comprising the steps of: a) selecting and/or isolating, from a heterogeneous group of MSCs, MSCs having top 30% expression level of neuron-glial antigen 2 (NG2); b) expanding the MSCs selected in step a), wherein the expanded MSCs having CD264+ are enriched; c) attaching the MSCs expanded in step b) to a scaffold; and d) implanting the scaffold with the MSCs in a mammal.
In embodiments, after the enrichment of CD264+ MSCs, at least 40% the expanded MSCs are CD264+. In embodiments, after the enrichment of CD264+ MSCs, at least 50% of the expanded MSCs are CD264+.
A method of making a scaffold having mesenchymal stem cells (MSCs) attached thereon is also described, comprising the steps of: a) selecting and/or isolating, from a heterogeneous group of MSCs, MSCs that do not express CD264; b) expanding the MSCs selected in step a), wherein the expanded MSCs having the bottom 50% expression level of NG2 are removed; and c) attaching the MSCs expanded in step b) to a scaffold.
In embodiments, in the expansion step b), the MSCs having the bottom 70% expression level of NG2 are removed.
As used herein, “scaffold” means a three dimensional structure that serves as a suitable support for the grown and proliferation of the stem cells, does not interfere with stem cell growth and viability, and permits adherence of the human mesenchymal stem cells. In embodiments, the scaffold is tricalcium phosphate/hydroxyapatite (HA/TCP) scaffold. In embodiments, the scaffold can be an elastomeric matrix that is preferably porous, and is reticulated and resiliently-compressible. For example, the elastomeric matrix can be made from a thermoplastic elastomer such as polycarbonate polyurethanes, polyether polyurethanes, polysiloxane polyurethanes, hydrocarbon polyurethanes, polyurethanes with mixed soft segments, and mixtures thereof, and preferably is made from polycarbonate polyurethane. It is also within the confines of the present disclosure that the matrix can be coated with a coating material such as collagen, fibronectin, elastin, hyaluronic acid or mixtures thereof to facilitate cellular ingrowth and proliferation.
As used herein, “high expression” refers to a expression level that is higher than the average expression level in any given group of heterogeneous cells. In one embodiment, “high expression” refers to the top 30% expression level among a group of cells. In one embodiment, “high expression” refers to the top 15% expression level among a group of cells. In one embodiment, “high expression” refers to the top 10% expression level among a group of cells. Genetically overexpressed NG2 can also lead to high expression of NG2 protein.
As used herein, “expression level” refers to the level of expression that can be used to sort the cells. There are several methods to measure the expression level of a surface marker, such as using the kinetics of antibody binding and radioactively labeled ligands, as well as using calibrated beads and flow cytometry, as known in the art. In embodiments, the expression level of NG2 or CD264 is measured using flow cytometry, and cells are sorted accordingly.
“Overexpression” or “overexpressed” is defined herein to be at least 150% of protein activity as compared with an appropriate control species or as having detectable expression of a gene not normally present in that host. Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, using highly active expression vectors, or upregulating the endogenous gene, and the like. An overexpressed gene can be represented by the + symbol, e.g., CD264+. In contrast, “expression” refers to normal levels of activity or better.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims or the specification means one or more than one, unless the context dictates otherwise.
The term “about” means the stated value plus or minus the margin of error of measurement or plus or minus 10% if no method of measurement is indicated.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or if the alternatives are mutually exclusive.
The terms “comprise”, “have”, “include” and “contain” (and their variants) are open-ended linking verbs and allow the addition of other elements when used in a claim.
The phrase “consisting of” is closed, and excludes all additional elements.
The phrase “consisting essentially of” excludes additional material elements, but allows the inclusions of non-material elements that do not substantially change the nature of the invention.
The following abbreviations are used herein:
In the invention disclosed herein, a novel method of selecting MSCs capable of long in vivo survival and preparing an implant by attaching the MSCs on a scaffold, with optionally removing CD264+ cells in order to improve the therapeutic efficacy. These changes result in MSC half-life that is among the longest reported in the literature. The method of this disclosure is based on the surprise findings that (1) NG2 expression level is higher on the longer surviving MSCs in vivo, (2) CD264+, while a marker for senescent MSCs, does not negatively impact MSC survival in vivo, and (3) CD264+ cells are less therapeutically effective.
Prior to this disclosure, colony forming efficiency in cell culture is accepted as a measure of in vitro cell survival, and extrapolated as an indicator of in vivo cell survival. However, the inventors discovered that CD264+ and CD264− MSCs have comparable in vivo survival kinetics. Such results prompted the need for a new method of screening for long in vivo survival MSCs in order to achieve better MSC therapies.
Inventors also discovered that NG2 expression level is elevated in those MSCs that survived longer in vivo, comparing to the MSCs that have shorter halflives. Further, while CD264+ MSCs exhibit similar in vivo survival results, their senescent status still make them less desirable for MSC therapies, and therefore negative selection of CD264+ cells would result in a group of MSCs that have both longer half-lives and better therapeutic efficacy.
For example, CD264 may be upregulated in aging hBM-MSCs as a potential stress response to facilitate cell survival. The upregulation of CD264 has been noted in several stress responses including ischemic preconditioning, oxidative stress, and inflammatory signaling. Previous work suggests that CD264 expression may have a prosurvival effect on cancer cells by mediating antiapoptotic signaling. In this context, CD264 may function to counteract the replicative stress of cellular aging in hBM-MSCs by promoting survival, as evidenced by the persistence of these cells following implantation.
The efficiency of the MSC attachment to the scaffold and the resultant in vivo retention of cells hold promise to develop reliable therapeutics. The Mastergraft product was chosen as a cellular scaffold due to its numerous clinical applications including spinal fusion, iliac crest backfilling, and dental surgery. Adapting the Mastergraft Mini Granules to these specific methods could generate clinic-ready bone grafts with improved chances for successful implantation. Additionally, this method can be extended to other implant materials and geometries to improve in vivo MSC retention. Graft materials in a block format have been explored in vivo and coral-based bone grafts are clinically used biocompatible scaffolds that exhibit similar properties to the Mastergraft granules. Using the attachment and preparation method detailed in this study to produce MSC-based constructs result in improved in vivo MSC survival for varying scaffold types compared to previous studies.
Furthermore, negative selection with CD264 to standardize MSC composition for implantation could produce more efficacious therapies. Previous work has shown that CD264+ MSCs are present in all MSC cultures, and this disclosure demonstrates that this aging population of cells has robust in vivo survival. It is well-established that aging and senescent MSCs have weakened regenerative potential in vitro and late-passage MSCs have been shown to be less effective in the treatment of graft-versus-host disease in humans compared to early-passage cells. Due to their in vivo persistence and poor regenerative properties, it is necessary to remove the aging CD264+ MSCs from the heterogenous culture using negative selection. The resulting MSC cultures should have robust regenerative properties resulting in improved therapeutic outcomes when implanted.
Negative selection to remove CD264+ MSCs may not always be a viable option due to a donor's high CD264+ content. Enrichment of these aging MSCs through positive selection could provide alternate treatment strategies. For example, the prolonged in vivo survival of aging CD264+ MSCs can be used to exploit effects of the senescence-associated secretory phenotype (SASP). The SASP is a result of cellular reprogramming during senescence where the bioactive molecules secreted by the cell drastically change. There is extensive literature detailing the potential therapeutic applications of the SASP for the treatment of liver fibrosis, wound healing, immune cell recruitment, and tissue regeneration. Creating an implantable construct containing aging CD264+ MSCs with predictable in vivo survival will be a reliable method to consistently deliver the beneficial SASP for a targeted application. Once the desired effect is achieved, the implant could be physically removed or the aging CD264+ MSCs targeted with a senolytic drug to clear the senescent cells. Additionally, positive selection using CD264 allows rejuvenation of the aging MSCs to restore their regenerative properties. If the desired outcome is integration of autologous MSCs into the target tissue, rejuvenating the aging CD264+ MSCs, such as through transient p38 inhibition or p53 inactivation, would be a necessary step to achieve an efficacious graft at the proper therapeutic dosage.
Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
This disclosure employed a well-established in vivo survival model that monitors bioluminescence from subcutaneous implants of GFP-FLuc MSCs on the dorsum of immunodeficient mice (
The in vivo results are also compared with in vitro results, particularly regarding the effect of NG2 expression level and CD264 expression on MSC survival. The comparison indicates that MSCs with high NG2 expression level tend to survive longer both in vitro and in vivo, whereas CD264+ and CD264− MSCs do not show significant difference.
Aging Phenotype of CD264-Sorted Populations
Cellular aging of MSCs was detected by expression of CD264. Heterogeneous cultures of transduced MSCs were 35% positive for CD264 expression based on a 1% isotype cutoff, consistent with the content of CD264+ cells that were observed for robust MSC cultures. P5 MSCs were sorted into aging CD264+ and control CD264− populations immediately prior to implantation to avoid artifacts in survival from differences in expansion and sorting conditions. Post-sort reanalysis confirmed distinct fluorescent separation between CD264+ and CD264− MSCs (data not shown). The colony-forming efficiency of each sorted population to be implanted was measured (
In Vitro Single-Cell Survival
Single-cell survival was comparable between CD264+ and CD264− MSCs when assessed by limiting dilution into 96-well plates for 7 days (
Nearly half of the single CD264− cells that survived on day 7 formed colonies ≥10 cells. Surviving CD264+ MSCs formed colonies less efficiency at ˜15% (p<0.05, n=3 replicates, 30-40 single cells/replicate,
Long term in vitro survival was also observed for 2 months (
Cell Attachment to Scaffold
Before implanting the cells into mice, MSCs were first attached to medical-grade HA/TCP granules (0.5 mm-1.6 mm particle diameter), a porous scaffold that is frequently used for ectopic MSC implants. Fluorescence from the transduced MSCs revealed the scaffold architecture of interconnected hollow shells with an outer shell diameter of 500 μm and inner pore diameter of 125 μm connecting the shells (
At this seeding density, all MSC preparations attached efficiently to the scaffold (
In Vivo Survival Kinetics
Bioluminescence imaging indicates that ectopic implants of aging CD264+ MSCs have similar survival kinetics to matched CD264− MSCs from the same culture (
CD264+ and CD264− implants from the same donor had comparable survival kinetics according to the parameters analyzed: rate of BLI signal decay, percent survival calculated from BLI signals during week 1 and 4, and signal half-life (
More specifically, the decay rate characterizes the exponential decrease in luminescence over time, and the Week 4 to Week 1 signal ratio estimates the percent of hBM-MSCs that survived after 1 month (data not shown). Signal half-life was determined for each sample to allow for intuitive interpretation of survival data and to facilitate meta-analysis across published in vivo MSC survival studies (
Perhaps most surprisingly, the mean half-lives in this example were among the highest values in the literature (
In contrast, there was significant donor-to-donor variation in the in vivo survival kinetics of MSC implants. MSC implants from the donor 2 survived longer with >2-fold difference in mean values of the kinetics parameters between the two donors (p<0.01, n=12 replicates per donor,
Different NG2 Expression Profiles in Donors
As a comparison, flow cytometric analysis revealed increased NG2 surface expression for high-survival hBM-MSCs from donor 2 relative to donor 1 hBM-MSCs (
Excised Implant
31 days after implant, the mice were sacrificed and the implants were excised to determine the hBM-MSC content thereof. The area fraction occupied by eGFP-positive cells in tissue sections (ROI fraction) was strongly correlated with the percent survival of implanted hBM-MSCs. It is found that the mean eGFP ROI fraction was 3× greater for the hBM-MSCs from donor 2 as compared with donor 1 (
Differentiation Potential
Specifically, the three upper-right datapoints came from MSC implants obtained from donor 2, whereas the three lower-left datapoints came from MSC implants obtained from donor 1. As discussed above regarding
Taken together, the similarity in the in vivo survival of CD264+ and CD264− implants in
In summary, while CD264+ has been reported as a marker for MSC senescence, the actual in vivo survival for CD264+ and CD264− MSCs shows no significant difference. On the other hand, not only is NG2 expression level an indicator of in vitro MSC proliferation and trilineage potential, its expression is also positively correlated with in vivo MSC survival and differentiation potential, which is important for MSC therapies. Therefore, NG2 is a good indicator for selecting MSCs with both long in vivo halflives and good differentiation potential for MSC therapies.
Human bone marrow MSCs will be collected from healthy donors at passage P3-P4, which is in the range of passage numbers for MSCs in clinical trial. The accepted criteria for human MSCs include: plastic-adherence, potency and immunophenotype. Only MSCs passing the criteria will be further cultured, and these properties will be reevaluated in MSC subsets and genetically modified MSCs.
The in vivo MSC survival will be assessed by bioluminescence imaging with a well-established humanized mouse model of ectopic implant survival. Briefly, human MSCs will be transduced with a lentivirus to express GFP and a luciferase. Implant (˜1 cm diameter) will consists of a fibrin gel containing 106 MSCs attached to 40 mg hydroxyapatite/β-tricalcium phosphate granules (˜1 mm diameter, Medtronic). Attachment of MSCs to the scaffold will be monitored by measuring DNA content extracted from the granules and verified by fluorescence microscopy to detect GFP expression by the transduced MSCs. The granules have a similar mineral composition to that of bone and are used as a bone void filler in oral and maxillofacial surgery. The MSC construct will be implanted subcutaneously on the dorsum of NIH III mice (Charles River Laboratories), which is a standard immunodeficient breed for xenoimplantation of human MSCs. Upon injecting the mice with luciferin, the bioluminescent signal from the implants will be quantified using the IVIS Lumina XRMS In Vivo Imaging System (PerkinElmer). With whole body scans, we verified that MSCs attached to the ceramic granules remain at the implant site. Also, we verified that the bioluminescent signal intensity correlates to the number of implanted GFP-positive MSCs.
Survival metrics: Using the survival model described above, each mouse will be implanted with a pair of NG2HI and NG2LO MSCs and an MSC-free control. We will inject the implanted mice with luciferin and measure the maximum radiance signal emitted by each implant every 3-5 days for a month. We will quantify the half-life of implanted MSCs (endpoint) defined as the (ln 2)/(radiance decay rate). The half-life is calculated using all the radiance data over 31 days and is preferred over the ratio of final-to-initial radiance (alternative), which is prone to larger error. We will validate our results with histological analysis of the area fraction of GFP+ MSCs within the implant after excision on day 31. Statistical analysis: Data acquisition and analysis will be blinded wherever possible. Differences in survival half-life between intra-mouse pairs of NG2HI and NG2LO MSCs will be analyzed with a mixed-effects ANOVA model to account for biological variation. A sample size n=7 donor pairs/donor sex will be required to detect differences among the groups based on 80% power, α=0.05, 2-fold difference in half-life and intra-mouse error of 25%.
It is expected to show that MSCs with high expression of NG2 will have significantly longer in vivo halflives as compared to MSCs having low expression of NG2.
Predicting in vivo MSC survival is practical in improving MSC therapies. To evaluate the ability of in vitro viability assays to predict in vivo MSC survival, modelling the association between in vivo MSC half-life and in vitro viability endpoints under ischemic stress by nutrient deprivation is proposed.
MSCs will be from randomly selected female and male donors. We will include any sorted MSC groups that exhibit a significant difference in implant survival.
In vivo survival assay: the MSC implant half-life is measured as discussed above.
Single cell survival assay: Single cells will be generated by limiting dilution into 96-well plates and detected by fluorescence microscopy. Endpoint is the percentage of single cells that survive after 7 days as measured by cell attachment.
Nutrient-deprivation assay: Constructs of luciferase-expressing MSCs will be prepared and attached to scaffold granules and encapsulated in a fibrin gel as described above. To mimic ischemia, MSC constructs will be maintained in hypoxic conditions in serum- and glucose-free medium. The O2 level in the in vitro construct will be measured with a needle microsensor (PreSens Precision Sensing) and will be controlled with an O2/N2/CO2 incubator (Thermo Fisher) to mimic the O2 level in the in vivo implant as described above. Under these conditions, it is reported that MSC viability declines steadily. Endpoint is the half-life of the bioluminescence from in vitro MSC constructs, which will be measured daily with our PerkinElmer Imaging System.
It is expected that a statistically significant association between in vitro and in vivo survival metrics for one or both in vitro assays as suggested by
The following methods were used in this disclosure.
MSC Cultures
Primary MSCs were isolated from iliac crest bone marrow aspirate from healthy adult volunteers with approval of the Tulane Institutional Review Board. Plastic-adherent MSCs prior to expansion were designated as passage 0 (P0). Donor MSC cultures employed in this study satisfy the criteria established by the International Society for Cellular Therapy for defining human MSCs based on plastic-adherence, immunophenotype and differentiation (Dominici et al., 2006). Unless otherwise noted, all cell culture supplies were obtained from Thermo Fisher Scientific (Waltham, Mass., USA). MSCs were routinely cultured in T-flasks using complete culture medium with antibiotics (CCMA): α-MEM with 2 mM L-glutamine supplemented with an additional 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 20% fetal bovine serum (FBS) (Sekiya et al., 2002). Cultures were inoculated at ≥100 cells/cm2 and maintained at 37° C. and 5% CO2 in a humidified incubator. Medium was completely exchanged every 3-4 days. At 50% confluence, cultures were subcultured using 0.25% trypsin/1 mM EDTA.
Other Cultures
COLO205 (ATCC CCL-222, Manassas, Va., USA) and G361 (ATCC CRL-1424) human cell lines were used for positive controls for cell surface expression of CD264 and NG2, respectively (data not shown). These cells were cultured according to supplier's instructions.
Lentiviral Transduction of MSCs
MSCs were transduced using either copGFP Lentiviral Particles (Santa Cruz Biotechnoloy, Dallas, Tex., USA) to express a bright GFP variant (GFP MSCs) or with RediFect Red Fluc-GFP Lentiviral Particles (PerkinElemer, Waltham, Mass., USA) to express red-shifted Luciola Italica luciferase fused by a T2A self-cleaving linker peptide to enhanced GFP (GFP-FLuc MSCs). P2 MSC cultures were inoculated at 1000 cells/cm2, and CCMA was replaced 24 h later with transduction medium: 100 μg/ml protamine sulfate (Sigma Aldrich, St. Louis, Mo., USA) in complete culture media containing no antibiotics (CCM). Medium volume was half of that for routine cultivation to promote transduction. Cultures were infected at a MOI of 20-25 and gently rocked a few times to evenly distribute viral particles over the cells (Lin et al., 2012). Spent medium was replaced with fresh transduction medium after 24 h, and a second dose of viral particles at the same MOI was added. Medium was replaced the following day with fresh CCM at standard volume. After 3 days, GFP-positive cells were collected by fluorescence-activated cell sorting (FACS) and cultured in CCMA until cryopreserved at passage 3.
Flow Cytometry
MSC cultures were amplified to P5 prior to flow cytometric analysis and FACS. Antibodies to detect human CD264 (PE-conjugated, FAB633P) and NG2 (APC-conjugated, FAB2585A) were obtained from R&D Systems (Minneapolis, Minn., USA). Antibodies to detect standard MSC markers were acquired as previously described (Madsen et al., 2017). Following gentle trypsinization and deactivation with CCMA, MSCs were resuspended in PBS at 0.5-1×107 cells/ml. Cell suspensions were incubated with antibody at saturating conditions for 30 min in the dark and on ice. Labeled cells were washed with 1×PBS and 1×4% FBS in PBS, and then resuspended at 2.5×106 cells/ml in chilled 4% FBS in PBS for analysis and sorting.
Flow cytometry was performed with a BD FACSAria Fusion flow cytometer equipped with FACSDiva software (version 8.0.1, BD Biosciences, Franklin Lakes, N.J., USA). Transduced MSCs were analyzed and sorted in tandem with matched isotype and mock-infected controls. Spectral overlap was corrected with multicolor compensation. Samples were gated to eliminate cellular debris and exclude doublets. MSCs were labeled with Fixable Viability Stain 780 (BD Biosciences) to assess viability, which was ≥90%. CD264− and CD264+ populations were sorted by capturing in purity mode MSCs with the bottom and top 10% of PE fluorescence, respectively. Aliquots of sorted cells were reanalyzed for PE fluorescence to validate sort purity. MSCs were sorted into chilled CCMA and then allowed to recover in T-flasks containing CCMA for 36 h prior to further experimentation.
Post hoc flow cytometric analysis was done with Kaluza software (version 1.3, Beckman Coulter, Brea, Calif., USA). MSCs with fluorescence greater than the 99th percentile of the fluorescence distribution for the isotype control were designated positive for antigen expression. Mean fluorescent intensity (MFI) ratios were reported as the MFI for the labeled sample relative to that of the isotype control.
Construct Preparation
Each construct was prepared with 40 mg of Mastergraft Mini Granules (15% hydroxyapatite/85% β-tricalcium phosphate, Medtronic, Memphis, Tenn., USA) (aliquoted into 50 ml vented conical tubes (CELLTREAT, Pepperell, Mass., USA). Granules were washed with 1×PBS and 1×CCMA, and then stored in 10 ml of CCMA overnight. After removing the medium, granules were seeded with 1×106 MSCs in 1 ml of prewarmed CCMA. Cells were mixed with the granules at 50-60 rpm for 6 h at 37° C. in a CO2 incubator. The MSC construct was centrifuged at 1000×g for 8 minutes, and supernatant was removed. Cell attachment was quantified by measuring (1) cells remaining in solution and (2) DNA content on the granules using the PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). To bind granules together, 15 μl of mouse fibrinogen (3.2 mg/ml in PBS, Oxford Biomedical Research, Rochester Hills, Mich.) and 15 μl of mouse thrombin (25 U/ml in 2% CaCl2, Oxford Biomedical Research) were added to each construct and allowed to coagulate for 1 min in a CO2 incubator (Mankani et al., 2008). After 1 ml of fresh CCMA was added to each tube, the construct was implanted.
In Vivo Survival Assay
Two- to four-month-old male and female NIH-III nude homozygous mice (Charles River Laboratories, Wilmington, Mass., USA) were implanted with MSC constructs with approval of Tulane's Institutional Animal Care and Use Committee. The animals were fed defined Purina LabDiet 5V5R (St. Louis, Mo., USA) starting 2 weeks prior to surgery and throughout the assay. Mice were anesthetized using isofluorane (MWI Animal Health, Boise, Id., USA) and administered 5 mg/kg Meloxicam (MWI Animal Health) subcutaneously prior to surgery. Each mouse was implanted with 3 MSC constructs: (1) CD264−, (2) CD264+, and (3) a cell-free control. Small incisions (1-2 cm) were made on the dorsal skin surface, and a subcutaneous pocket was created by blunt dissection. A single construct was inserted into each pocket. Incisions were closed with simple interrupted sutures and covered with Vetbond Tissue Adhesive (3M, Maplewood, Minn., USA). The mice were examined with bioluminescence imaging over 31 days and then humanely sacrificed using CO2 asphyxiation followed by cervical dislocation.
In Vitro Survival Assays
MSCs were stained with 10 μM CellTracker Green (Thermo Fisher Scientific) and plated by limited dilution into 96-well plates. Wells containing a single cell were detected by fluorescence microscopy. Cells were restained with CellTracker Green after 3 days and with crystal violet (Sigma Aldrich) after 1 week to identify single cells that survived and formed colonies (≥10 cells). In vitro survival of MSC constructs was monitored with bioluminescence imaging. Constructs were cultured in 50 ml vented conical tubes with constant mixing at 50-60 RPM and complete medium exchange every 2-3 days. Every 2 weeks for 2 months, the constructs were transferred to 24-well plates containing CCMA for bioluminescence imaging.
Bioluminescence Imaging
Bioluminescence of cell cultures and implants was measured using an IVIS Lumina XRMS In Vivo Imaging System (PerkinElmer) with Living Image software (version 4.4, PerkinElmer). For in vivo imaging, mice were sedated with isofluorane and 100 μl Xenolight D-Luciferin (30 mg/ml, PerkinElmer) was administered subcutaneously adjacent to each implant. For in vitro imaging, MSC constructs were exposed to 300 μg/ml D-luciferin in CCMA. Bioluminescence was acquired every 5 min after luciferin addition using the automatic exposure settings until the bioluminescent signal decreased. Maximum radiance in the region of interest around each construct was measured every 3-4 days for 31 days and background corrected. When grouped together, representative bioluminescent images were placed on an identical radiance color scale. Radiance data were natural log-transformed, and a linear regression was performed. The slope of the regression line corresponds to the rate of radiance decay. Signal half-life (t1/2) was calculated from the decay rate (λ) using the following formula:
Other Assays
Colony-forming efficiency was evaluated according to Barrilleaux et al. (2009). MSCs were plated at a clonogenic level of 100±10 cells in a 10 cm cell culture dish with 15 ml CCMA. Samples were cultured undisturbed for 14 days and then stained with crystal violet to detect cell colonies (≥50 cells). Senescence-associated β-galactosidase (SA β-Gal) activity at pH 6.0 was assessed in subconfluent MSC cultures using Senescence Cells Histochemical Staining Kit (Sigma Aldrich). MSCs stained at pH 5.0 served as a positive β-Gal control. Osteo-, adipo-, and chondrogenesis were induced in MSCs and evaluated after 21 days of differentiation. Alizarin Red S (Sigma Aldrich) detected calcified extracellular matrix in osteogenic samples, AdipoRed (Lonza, Walkersville, Md., USA) stained lipid droplets in adipogenic cells, and Alcian Blue (Sigma Aldrich) identified matrix deposition of sulfated glycosaminoglycans during chondrogenesis.
The following references are incorporated by reference in their entirety for all purposes.
This invention claims priority to U.S. 62/820,367, filed on Mar. 19, 2019 and incorporated by reference in its entirety herein for all purposes.
This invention was made, in part, with support provided by the United States government under Grant Nos. CBET-1066167 and CBET-1604129 awarded by the National Science Foundation. The Government has certain rights in this invention.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/023459 | 3/18/2020 | WO | 00 |
Number | Date | Country | |
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62820367 | Mar 2019 | US |