Stem Cell Analysis

Overview of Stem Cell Analysis

Stem Cells:
- Definition: Cells with the unique ability to self-renew and differentiate into specialized cell types
- Types:
  - Hematopoietic Stem Cells (HSCs): Give rise to all blood cell types
  - Mesenchymal Stem Cells (MSCs): Give rise to bone, cartilage, fat, and other connective tissues
  - Embryonic Stem Cells (ESCs): Pluripotent cells derived from the inner cell mass of the blastocyst
  - Induced Pluripotent Stem Cells (iPSCs): Adult cells that have been reprogrammed to become pluripotent
Importance of Stem Cell Analysis:
- Transplantation: Assessing the quality and quantity of stem cells for transplantation
- Regenerative Medicine: Studying stem cell differentiation and function for regenerative medicine applications
- Disease Modeling: Using stem cells to model and study diseases
- Drug Discovery: Using stem cells to screen for new drugs

CD34 Absolute Counts

CD34:
- Definition: A cell surface glycoprotein that is expressed on hematopoietic stem cells (HSCs) and progenitor cells
- Importance: CD34 is a commonly used marker to identify and quantify HSCs
CD34 Absolute Count:
- Definition: The number of CD34+ cells per unit volume of blood or bone marrow
- Clinical Significance:
  - Predicting engraftment after stem cell transplantation
  - Monitoring stem cell mobilization
  - Diagnosing and classifying hematologic disorders
Methods for CD34 Absolute Counts:
- Single-Platform Flow Cytometry:
  - Principle: Uses a single flow cytometer to simultaneously measure the number of CD34+ cells and the total white blood cell count
  - Advantages: More accurate and precise than dual-platform methods
- Dual-Platform Flow Cytometry:
  - Principle: Uses a flow cytometer to measure the percentage of CD34+ cells and a hematology analyzer to measure the total white blood cell count
  - Advantages: Less expensive than single-platform methods
  - Disadvantages: Less accurate and precise than single-platform methods

Single-Platform Flow Cytometry for CD34 Absolute Counts

Principle:
- A known volume of sample is stained with antibodies against CD34 and other cell surface markers
- A known number of fluorescent beads is added to the sample as an internal control
- The sample is acquired on a flow cytometer, and the number of CD34+ cells and beads is counted
- The absolute count of CD34+ cells is calculated using the following formula:
  - CD34+ cells/μL = (Number of CD34+ cells / Number of beads) x (Number of beads added / Sample volume in μL)
Procedure:
1. Sample Preparation:
  - Collect blood or bone marrow aspirate in EDTA anticoagulant
  - Lyse red blood cells (RBCs) using a lysing buffer
  - Wash the sample to remove excess lysing buffer
2. Antibody Staining:
  - Incubate the sample with fluorochrome-conjugated antibodies against CD34 and other cell surface markers (e.g., CD45)
  - Include appropriate isotype controls
3. Bead Addition:
  - Add a known number of fluorescent beads to the sample
4. Flow Cytometry Acquisition:
  - Acquire the sample on a flow cytometer, following the manufacturer’s instructions
  - Collect a sufficient number of events to ensure accurate counting
5. Data Analysis:
  - Gate on the CD45+ leukocyte population
  - Identify the CD34+ cell population within the CD45+ gate
  - Count the number of CD34+ cells and beads
  - Calculate the absolute count of CD34+ cells using the formula above
Reagents and supplies:
- CD45-FITC: to gate on leukocytes
- CD34-PE: to determine the percent of hematopoietic stem cells
- 7-AAD: to remove any dead cells from the analysis
- Counting beads: to determine the absolute counts

Gating Strategy for CD34 Absolute Counts

Gate on the Leukocyte Population:
- Use CD45 and side scatter (SSC) to identify the leukocyte population
- Exclude red blood cells and debris
Gate on the CD34+ Population:
- Use CD34 and side scatter (SSC) to identify the CD34+ cell population
- Exclude non-specific binding using isotype controls
Confirm CD34+ Population:
- Use CD34 and CD45 to identify true CD34+ cells from the background noise
Bead Gate:
- Use forward scatter (FSC) and side scatter (SSC) to identify the beads population to count how many events are being tested.

Controls for CD34 Absolute Counts

Isotype Controls:
- Used to assess non-specific antibody binding
Negative Controls:
- Used to set the baseline for CD34 expression
Positive Controls:
- Used to validate the staining protocol and instrument performance
Beads Controls:
- Used to make sure that the beads are distinguishable from any debris that may be present in the sample
Automated gating:
- It is imperative that the gating is not automated to prevent the omission of a small CD34+ cell population

Clinical Applications of CD34 Absolute Counts

Stem Cell Transplantation:
- Predicting engraftment after autologous or allogeneic stem cell transplantation
- Monitoring stem cell mobilization prior to collection
- Assessing the quality of stem cell products
Hematologic Disorders:
- Diagnosing and classifying acute leukemias
- Monitoring minimal residual disease (MRD) in acute leukemias
- Assessing bone marrow failure syndromes
Other Applications:
- Monitoring immune reconstitution after chemotherapy
- Studying stem cell biology and differentiation

Other Stem Cell Markers

CD133 (Prominin-1):
- Another marker of HSCs and progenitor cells
- Often used in combination with CD34
CD90 (Thy-1):
- A marker of mesenchymal stem cells (MSCs)
STRO-1:
- Another marker of MSCs
Oct4, Nanog, Sox2:
- Transcription factors that are expressed in pluripotent stem cells (ESCs and iPSCs)

Troubleshooting Stem Cell Analysis Assays

Low CD34+ Cell Count:
- Possible Causes:
  - Sample degradation
  - Incorrect staining
  - Instrument malfunction
- Troubleshooting Steps:
  - Use fresh samples
  - Verify staining protocol
  - Inspect instrument for malfunctions
High Background Noise:
- Possible Causes:
  - Non-specific antibody binding
  - Autofluorescence
  - Contamination
- Troubleshooting Steps:
  - Use blocking reagents
  - Reduce autofluorescence
  - Clean samples
Variable Results:
- Possible Causes:
  - Inconsistent technique
  - Instrument instability
  - Sample heterogeneity
- Troubleshooting Steps:
  - Standardize assay protocols
  - Calibrate instrument
  - Increase sample size

Key Terms

Stem Cell: A cell with the ability to self-renew and differentiate into specialized cell types
Hematopoietic Stem Cell (HSC): A stem cell that gives rise to all blood cell types
CD34: A cell surface glycoprotein that is expressed on HSCs and progenitor cells
Absolute Count: The number of cells per unit volume
Single-Platform Flow Cytometry: A method for simultaneously measuring the number of CD34+ cells and the total white blood cell count
Isotype Control: An antibody that is used to assess non-specific antibody binding

Solid Organ Transplant

Cell Cycle