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A microfluidics platform for rapid and direct enumeration of rare stem…

- - - - Cell cycle
      - Cell lineages :star:
      - Other stochastic cellular processes
      - Response to stimuli
    - - Viral infection
      - Stem cells :star:
      - Genetic abnormalities
      - Fetal cells in maternal blood
      - Minimal residual disease (CTCs, CSCs)
  - - - High sensitivity
        
        High signal-to-noise ratio
      - High throughput
        
        High capture rate
        
        Rapid/parallel sample generation
      - High sample quality (limit changes in gene expression, degradation or bias)
        
        Short processing time
        
        No fixation steps
        
        Spatial, morphological information
        
        Gentle dissociation procedure
        
        Min contamination
        
        Cell capture rate/starting cell number
    - - Versatile
        
        Easy to optimize for other markers
      - Quick turnaround
        
        Complexity (potocol & analysis)
      - Easy to use
        
        Complexity
        
        Expertise
      - Automatable
        
        Complexity
      - Cost-effective
        
        Complexity (instrument)
        
        Reagents
      - Multiplex capability
        
        Number of targets
        
        Multianalyte analysis
  - - - Epigenome
        
        Chromatin structure
        
        ChIP-seq
        
        DNA methylation
        
        scRRBS
        
        Methyl-seq
        
        Chromatin accessibility
        
        ATAC-seq
      - Metabolome
        
        Matrix-Assisted Laser Desorption Ionization (MALDI)
        
        Live single-cell mass spectrometry (LSCMS)
      - Proteome
        
        Immunoassays + high-content screening
        
        Flow cytometry/FACS
        
        Mass flow
      - Transcriptome
        
        FISH
        
        Nanostring
        
        RNA sequencing
        
        RNA microarray
        
        RT-PCR :star:
      - Genome
        
        FISH
        
        PCR
        
        Sequencing
        
        MDA
        
        MALBAC
    - - Mouth pipetting
      - Flow cytometry
      - Microwell
      - Microdroplet
      - Laser capture microdissection
      - DEPArray
      - Microcircuits/chambers
      - Micromanipulation
      - Laser capture microdissection (LCM)
- - - - Self-renewal
      - Generates all blood lineages
        
        What are they?
    - - Transplantation treatments
        
        Ex vivo expansion
        
        Derivation from PSCs
        
        Clinical measurements
      - Developmental biology
    - - LT (permanent)
      - IT (6-8 months)
      - ST (4-6 weeks)
  - - - Surface markers
        
        Lin-Sca1+ (0.5% BM)
        
        Lin-
        
        CD45R/B220, CD19=B-cells
        
        CD5/Ly-1=Lymphocytes
        
        CD11b/Mac-1=Macrophages
        
        Ly-6G/Gr-1=Granulocytes
        
        Ter-119/Ly-76=Erythrocytes
        
        CD8a/Ly-2, CD4/Ly-4=T-cells
        
        Lin-Sca1+c-Kit+ (LSK) (0.05% BM)
        
        Contains all HSCs
        
        LKS Thy1.1-lo
        
        contains all LT-HSCs
        
        LSK CD34- (0.005% BM)
        
        contains all LT-HSCs
        
        LSK CD34- FLK2/Flt3-
        
        LSK CD34- FLK2+ = ST-HSC
        
        CD150/SLAMF1 + CD244 − CD48- (SLAM)
        
        EPCR SLAM
        
        RKSL CD34-lo FLK2/Flt3- CD49lo
        
        Lin- Mac-1- CD4 -
        
        contains all LT-HSCs
        
        LSKR CD49b-lo CD150-hi
        
        close to purity, 50% reconstitution in competitive assay = 1 in 20,000 BM cells?
        
        ~20% LSKR
        
        LSKR CD49b-lo
        
        30% functional purity
        
        <3% of LSK cells
      - Dye exclusion
        
        Hoechst 33342-lo
        
        Rho-lo
        
        selective for quiescent cells
    - - Competitive
      - Limiting dilutions
      - Serial transplantation
  - - - IL7R-a
      - EPCR
      - Gata3
        
        In LT-HSC but not IT. Expressed in some mature cells
    - - EPCR
      - IL7Ra
      - Gata3
        
        Constrains LT-HSC proliferation
        
        T cell development