Towards Improved Characterization Of MSCs
Developmental StagesEnvironmental InfluencesTowards ImprovedCharacterization of ionSteven R. Bauer, Ph.D.Cell and Tissue Therapy BranchDivision of Cell and Gene Therapy14Potential ForMSC-BasedRegenerativeMedicine Products:What do youmeasure to identifythe effective cells?ClinicalResponseTotal olRegenerateIdentification and correlation of MSC attributes within vivo and in vitro assays of safety and efficacy:CBER/FDA MSC consortiumMcCright Lab: in vivo model ofMSCsMultipotent Stromal CellCharacterizationfatothermuscleboneAlterman Lab: proteomicsCytoplasm33%rare cellAdipose tissue l hind limb ischemiaCorrelatecandidateattributeswith assayoutcomesMoos Lab: geneexpression,qRT-PCR,single cell PCRSources:Bone marrow (MSC)abundant cellPuri Lab: genomicsProduct characteristicsstromaClinical Uses:RegenerativeCardiac repairBone repairImmunomodulatoryGVHDactiveBauer Lab: in vitroquantitative proliferationand differentiationadipogenesisWei Lab: in vitro,in vivoimmunosuppressionHursh lab:epigenetics,karyotypesmT-cell61
Cell Size Increases with TissueCulture Passage: Donor DifferencesMSC ManufacturingGoal: 8 donors Expand to 100 T175 flasks Change media every third day Expand to 80% confluence Harvest at p3, p5, p7 Cryopreserve, snap freeze, orinitiate analytical proceduresPassage 325Passage 5Ave rage Cell Diameter (microns)Passage 7Yield 2-3 x 108 cells per 6601278F35M1662PCBM1641696B167PC10CFU-F Activity Decreases with TissueCulture Passage: Donor Differences Publicly available sourcesSingle method, single lab, uniform media Sufficient MSCs for multiple characterization approachesOne lot of FCS60Passage 3Passage 5% Colony Forming Units 8777MSC Production 110PCBJess Lo SurdoEva RudikoffM16320Passage 110PCBM163208Source: ALLCELLS,Cell Size Distribution Patterns Changeswith Tissue Culture PassagePCBM1632 P3 Size Distribution (n 13)Average 14.70umAverageDiameterSize 14.70um302010Average FrequencyAverage FrequencyPCBM1641 P3 Size Distribution (n 13)5040080706050403020100Cell Size (um)6040Average 15.97umAverageDiameterSize 15.97um302010MSC0Cell Size (um)PCBM1641 P7 Size Distribution (n 15)Average Diameter 16.28umAverage Size 16.28umCell Size (um)PCBM1632 P7 Size Distribution (n 5)Average FrequencyAverage FrequencyOsteogenesis50Cell Size (um)35302520151050(-) CONTROLPCBM1632 P5 Size Distribution (n 13)Average FrequencyAverage FrequencyAverageDiameterSize 15.45umAverage 15.45umAverage Size 14.41umAverage Diameter 14.41umINDUCTION ationCell Size (um)PCBM1641 P5 Size Distribution (n Diameter19.67umAverageSize 19.67um912Cell Size (um)2
Automated Microscopy For AdipogenesisLimiting Dilution AssayP31001106789 10 11 12ABCDEFHP5exponential form: y AeBxy 0.37x precursor frequencyP70.10.0116/247/24 Adipogenic stimulationCell Dose2/24 Undifferentiated controlTo exclude edge effects, outer 15% of well was not sampled25 images per wellNile Red positive cells associated with nuclei counted and normalizedto total nuclei count per imagen 3/Donor/passageImaging 4% of specified area at 10X responding wells Lo Surdo, JL, and Bauer, SR. Quantitative Approaches to Detect Donor and PassageDifferences in Adipogenic Potential and Clonogenicity in Human Bone Marrow-DerivedMesenchymal Stem Cells. 2012. Tissue Engineering: Part C 18: 877-889. Adipogenic Precursors Can Changewith Tissue Culture PassageAPassage 70.80.60.40.201256302004000.0100P5 1/1260.0010P5 12 in 126P3P3 1 1/125in 1250.0001Passage 51Passage 70.80.60.40.20125633210Fraction of Non-RespondingWellsFraction of Responding WellsPCBM1641 Precursor FrequencyPassage 3250Passage 5Passage 7PCBM1641 Limiting Dilution (n 3)Cell Dose (cells/well)Passage 3Cell Dose (cells/well)1.2500R 0.86R2 0.930.000032Processed for Counting8001000P7 1P7 in 2,4441/2444R2 0.96Cell Dose 0Passage 3Passage 50.01Passage 70.001P7 1/73P3 R12 in0.99820.0001 1 in 70P5 P5 1/700.00001R2 0.96Nuclei/ Fat /Nuclei with FatDAPI (DNA)Nile Red (intracellular fat)P7 1 in 73Cell Dose (cells/well)14P3 1/8217R2 0.99Adipogenic Precursors areAssociated with Smaller MSCsAdipogenic Activity Decreases withTissue Culture Passage: DonorDifferences16Passage 314Passage 5Passage 712Large cells l cells 1/138P 0.02415*P 0.05**P 0.01***P 0.001****P 0.0001Relative to P3127756250Image1.0000% Nile Red Positive500Fraction of Non-RespondingWellsFraction of Responding WellsPassage 51B10.0000Passage 31000Automated Microscopy forAdipogenesisPCBM1632 Precursor FrequencyPCBM1632 Limiting Dilution (n 3)1.216Lo Surdo JL et al., Cytotherapy, 2013. In 6625PCBM1641416769631108772PCBM163210001Fraction of Non‐responding WellsCell Dose:(cells/well)183
Consensus MSC Markers do not Correlate withFunctional Heterogeneity, Donor or TissueCulture Age Differences% Positive% Positive% Positve1001009090805805Passage 3Passage 5Passage 5167696PCBM1641Passage 7 110877 PCBM16628F35600Passage 3Passage 7PCBM16329080500 CD73CD44CD29100MSC ImmunosuppresionPassage 3Passage 5Important attribute for allogeneic useMeasured by cell-based assaysUse of human cells T-cells contributes tovariability of assay read out127756Passage 7PCBM1655CD105CD90% Positive9090805805Passage 3Passage 5900Passage 3Passage 7Passage 5Passage 7Can mouse T-cell functions be suppressed byhuman MSCs? 80500 100% Positive100100% PositiveCD166Passage 3Passage 5Passage 7International Society for Cell Therapy: Cytotherapy (2006) Vol. 8, No. 4, 315-317. MSCs are characterized asbeing adherent to plastic, capable of tri-lineage differentiation, 95% of the MSC population must express19CD105, CD73 and CD90, as measured by flow cytometry. Additionally, these cells must lack expression( /2%positive) of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA class II.Use T-cell receptor transgenic mice All T-cells express same antigen receptor Known antigen No genetic variability22NOD/ShiLtj 1632167696PCBM16411. Proteomic characterization schemeCell culturing, protein harvesting using variousplatforms, and proteome fractionation80% confluent mesenchymal stromal cells Proteome harvestingProteome fractionation2D NanoLC/2D NanoUPLCMass spectrometry-based protein identification,quantitation, expression dynamic profiling MSCconcentrationdependentMSC specific Proteome coverage (7753 proteins in total)LTQ, ThermoMALDI‐TT, ABSciex Synapt G2, WatersTandem 2%156121%ESI Ongoing 230631%MALDIHepG2hepatocarcinomaHT-180 epitheliallineApply to all donors,all passages23202. Overview of proteome summary and comparison between donorsI. Proteome functional Class summaryModulation of surface activation markers on murine T cells by human MSCs,as determined by mean fluorescence intensity (MFI)II. Shared proteins between donorsDevelopment,8%CD4 T cellsA350050000300040000Cell cycle, 5%CD25CD8 T cellsB600003000025002000150020000Immunity, 5%1000100005000Apoptosis, 025002000200015001000III. Proteomic variability between donorsDonor IDPCBM164128.41676968.7PCBM163223.3110877500% 00003000070000250006000020000Average of 21%IV. Comparison between donors based on 2000250010000200080001500CD62L6000% variability: ratio of proteome size of a donor ascompared to proteome size constructed using21proteins expressed in at least 3 donors10004000200005000T cellsT cells APCs peptideT cells APCs peptide MSCs (10:1)T cells APCs peptide MSCs (5:1)244
Significance for Cell Therapy Quantitative functional assays; a crucial part of asystematic approach to identify and qualify predictiveproduct characteristics for cell therapy products Consensus MSC Markers do not Correlate with FunctionalHeterogeneity: Donor or Tissue Culture Age DifferencesRobust enumeration of functional sub populations illustratesimportance of extensive single cell analysesAssess Differences between MSC donors Impact of tissue culture conditions and duration Correlation with other characteristics of MSCs Enrichment techniquesApplication to understanding mechanisms controllingstem-cell differentiation and function HeterogeneityImproved understanding of “stemness”Mechanisms of archIn-processand releasePtestsgeneexpressionThe Rosetta StoneCritical ements Bauer Lab Jessica Lo SurdoEva Rudikoff MSC Consortium Funding FDA Modernizing ScienceInitiativeFDA MCMiBARDACBER/OCTGT/DCGT Michail AltermanDeb HurshBrent McCrightMalcolm MoosRaj PuriCheng-Hong Wei275
Differences in Adipogenic Potential and Clonogenicity in Human Bone Marrow-Derived Mesenchymal Stem Cells. 2012. Tissue Engineering: Part C 18: 877-889. 14 PCBM1632 Precursor Frequency 0.0000 0.0001 0.0010 0.0100 0.1000 1.0000 10.0000 0 200 400 600 800 1000 Cell Dose (cells/well) Fraction of Non-Responding Wells Passage 3 Passage 5 Passage 7
Enable vSphere HA and vSphere DRS in a Cluster (MSCS) 29 Create VM-VM Affinity Rules for MSCS Virtual Machines 29 Enable Strict Enforcement of Affinity Rules (MSCS) 30 . This chapter includes the following topics: . n Setting up Clustered Continuous Replication or Database Availability Groups with Exchange n Setting up AlwaysOn Availability .
The MSCS program sheet The central requirement for the MSCS degree is completion of at least 45 units that represent an approved academic plan. The concrete representation of that academic plan is your program sheet, which lists the courses you i
of SFM-expanded MSCs [19,20]. However, the safety and efficacy of MSCs cultured in SFM have not been well eval-uated [21]. In this study we investigated whether human umbilical cord mesenchymal stem cells (hUC-MSCs) ex-panded in SFM change their biological characteristics and
Multiple Sclerosis Rehabilitation Interventions Across the Spectrum of Disability and Continuum of Age Marissa Barrera, MS, MPhil, MSCS, TSHH, CCC-SLP Evan Cohen, PT, MA, PhD, NCS Linda Csiza, PT, DSc, NCS Jennifer Kalina, MS, OTR/L, CCRC, MSCS Herb Karpatkin, PT, DSc, NCS, MSCS Stephen Krieger, MD Patient Management Across Levels of Disability
Characterization: Characterization is the process by which the writer reveals the personality of a character. The personality is revealed through direct and indirect characterization. Direct characterization is what the protagonist says and does and what the narrator implies. Indirect characterization is what other characters say about the
cells by their adherent nature in in vitro cell cultures and fibroblastic morphology [6, 7]. The function of BMMSCs was initially thought to be limited to suppor-ting hematopoiesis; indeed, one of the first clinical use of these progenitor/stem cells was to enhance HSC engraftment [8]. Since these early reports, MSCs have
characterization: direct characterization and indirect characterization. Direct Characterization If a writer tells you what a character is like the method is . Dr. Chang was the best dentist in the practice. He had a charming smile, a gentle manner, and a warm personality.
our characterization. Given this novel characterization, we can pro-duce models that predict optimization sequences that out-perform sequences predicted by models using other characterization tech-niques. We also experimented with other graph-based IRs for pro-gram characterization, and we present these results in Section 5.3.
