Podcast
Questions and Answers
What is the primary purpose of the transplantation assay mentioned?
What is the primary purpose of the transplantation assay mentioned?
How can donor cells be distinguished from recipient cells in the assay?
How can donor cells be distinguished from recipient cells in the assay?
What does flow cytometry visualize in the context of the assay?
What does flow cytometry visualize in the context of the assay?
What feature allows tracking of donor stem cells in the recipient mouse?
What feature allows tracking of donor stem cells in the recipient mouse?
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For donor stem cells to be confirmed as true stem cells, what must they accomplish?
For donor stem cells to be confirmed as true stem cells, what must they accomplish?
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Which cell type is indicated as a marker for HSC activity?
Which cell type is indicated as a marker for HSC activity?
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What type of cells are specifically analyzed in the transplantation assay?
What type of cells are specifically analyzed in the transplantation assay?
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What methodology is essential for collecting data about donor cells post-transplantation?
What methodology is essential for collecting data about donor cells post-transplantation?
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What is the main purpose of using fluorescent reporters in lineage tracing?
What is the main purpose of using fluorescent reporters in lineage tracing?
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Which gene is mentioned as being part of the reporters used for HSC-specific expression?
Which gene is mentioned as being part of the reporters used for HSC-specific expression?
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What mechanism allows the CRE to become active in the presence of tamoxifen?
What mechanism allows the CRE to become active in the presence of tamoxifen?
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Which of the following genes is associated with the Tie2 system in HSC lineage tracing?
Which of the following genes is associated with the Tie2 system in HSC lineage tracing?
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What type of genetic component is utilized in the system for expressing fluorescent reporters?
What type of genetic component is utilized in the system for expressing fluorescent reporters?
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The presence of which compound is essential for the activity of CreERT2?
The presence of which compound is essential for the activity of CreERT2?
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What happens to the expression of the fluorescent reporter once activated in the context of lineage tracing?
What happens to the expression of the fluorescent reporter once activated in the context of lineage tracing?
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What role does tamoxifen play in the use of genetic elements for lineage tracing?
What role does tamoxifen play in the use of genetic elements for lineage tracing?
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What does the variation in lineage output during transplantation indicate about hematopoietic stem cells (HSCs)?
What does the variation in lineage output during transplantation indicate about hematopoietic stem cells (HSCs)?
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What does the phrase 'deficiency in production of one or more lineages' refer to?
What does the phrase 'deficiency in production of one or more lineages' refer to?
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Which statement best describes what transplantation can reveal about HSCs?
Which statement best describes what transplantation can reveal about HSCs?
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In the context of HSC lineages, what does myeloid vs lymphoid bias signify?
In the context of HSC lineages, what does myeloid vs lymphoid bias signify?
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What is NOT implied by having different lineage outputs from HSCs during transplantation?
What is NOT implied by having different lineage outputs from HSCs during transplantation?
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What role does steady state play in relation to HSC function?
What role does steady state play in relation to HSC function?
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Which of the following genes is associated with hematopoietic stem cells (HSCs)?
Which of the following genes is associated with hematopoietic stem cells (HSCs)?
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What does the color coding of pink and blue represent in relation to gene expression?
What does the color coding of pink and blue represent in relation to gene expression?
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Study Notes
Stem Cells and Cancer Research
- Images show microscopic views of stem cell cultures at different time points (Day 2, Day 9, Day 13).
- Research presentation by Katie Sawai from INSERM U1312 BRIC Team #11 on October 5, 2022.
What is a Stem Cell?
- Stem cells have the ability to self-renew, replicating themselves while maintaining the potential to differentiate into specialized cells.
- They also have the capacity to differentiate into the diverse cell types that make up a tissue.
What Defines Cell Identity?
- Cell identity is defined by the ability of cells to self-renew and differentiate.
- Regenerating and giving rise to specialized cells that make up a tissue.
Reprogramming of Somatic Cells
- Dolly the Sheep (1996) was cloned through the reprogramming of somatic (adult) cells.
- The process involved transferring the nucleus of an adult cell into an enucleated egg cell.
- This process suggests that factors in the recipient's oocyte can induce the stem cell state.
- Somatic cells contain the entire DNA required for full organism development.
- Reprogramming can transform a fully differentiated somatic cell into a stem-like state.
Inducing Pluripotency
- Mature cells can be reprogrammed to become pluripotent.
- Certain transcription factors (KLF4, SOX2, c-Myc, Nanog, Oct-3/4, LIN-28) are involved in the reprogramming of mature cells into induced pluripotent stem (iPS) cells.
Functional Studies of Stem Cells
- Techniques used to study stem cell function include flow cytometry & sorting, in vivo gene targeting, microarray/next-generation sequencing, colony assays, transplantation, and lineage tracing.
- These allow researchers to understand how stem cells behave and lead to differentiation.
Hematopoietic Stem Cell (HSC)
- Artur Pappenheim and Alexander Maximov were early researchers on blood cell development.
- HSCs give rise to all the different blood cell types.
- A common stem cell for different blood cells exists during embryonic development and throughout the life of mammals.
Stem Cell Pioneers (1960s)
- Ernest McCulloch and James Till were pioneers in understanding hematopoietic stem cells.
- Spleen colony assay (1961) for study of single cell-derived multilineage (erythroid/myeloid) transplantable cells capable of differentiation.
- Clonogenicity, pluripotency, and self-renewal were key concepts.
Can These Primitive Cells Be Grown In Vitro?
- This question investigates the possibility of growing primitive cells, like stem cells, outside the body in a laboratory setting.
In Vitro Culture Conditions
- Table 1 describes various media and factors for in vitro culture of mouse stem cells.
- Media conditions include granulocyte-macrophage colony stimulating factors (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF).
Caveats
- Early assays mainly show myeloid (white blood cell) lineage potential.
- Limited lymphoid (immune cell) lineage reading.
- Distinction between self-renewing and non-renewing progenitors needed.
- Ensuring stem cell properties is important.
- Defining the best phenotype for HSCs is vital.
- Flow cytometry is essential for detailed cell characterization and identification of specific markers for individual cells.
Technological Advance: Flow Cytometry
- Cell suspension travels through a flow chamber.
- Different lasers allow cells to be stained and observed for different markers.
- Enables researchers to rapidly characterize large numbers of cells based on their fluorescence profile, which is measured and recorded.
- Identifying various cell types within a mixture by analyzing expression levels of certain markers.
Transplantation
- Transplantation is a "gold standard" method for evaluating HSC function.
- Determining if donor cells engraft and function in a recipient animal measures functionality.
Prospective Identification of HSCs
- Prospective identification involves isolating HSCs based on specific cell surface markers and analysis of specific function, and identifying these through flow cytometry and other techniques for studying the lineage potential during development.
Identification of Progenitors
- Progenitor cells act as intermediate steps between HSCs and mature cells.
- Identification of progenitors for particular lineages is crucial for developing effective therapies. They contribute to steady-state hematopoiesis.
- Progenitors include common lymphoid and common myeloid progenitors.
Classical View of Hematopoiesis
- Diagram illustrating differentiation pathways.
- The various types of cells (Erythrocytes, Platelets, Granulocytes, Macrophages, Dendritic cells, T cells, NK cells, and B cells) emerge from common progenitors.
What Regulates HSC Function?
- Many genes are involved in regulating HSC function, including transcription factors, epigenetic factors, and cell cycle regulators.
Technological Advance: In Vivo Genome Modification
- Techniques for modifying an organism's genome by introducing, deleting, or changing sequences involve creating specific mutations to study the function of genes.
- Reporter modification (creating a readout of genes of interest) can be achieved.
- Methods like homologous recombination and random integration enable precise manipulation.
HSC-Specific Genes
- Discovery of genes uniquely expressed in HSCs helps understand their differentiation and how they maintain their identity.
- Expression levels of specific genes are higher in HSCs compared to other progenitors.
HSC-Specific Reporters
- Tools and systems for tracking the activity and behavior of HSC (using techniques for specific labeling).
- Gene promoters associated with HSCs can be used to track lineage development and lineage development of different cell types.
In Vivo Lineage Tracing: Fluorescent Reporters
- In vivo lineage tracing allows for the dynamic tracking of cell lineage.
- The method involves using fluorescent tags to label stem cells, allowing monitoring of their progeny as cells differentiate.
In Vivo Lineage Tracing of HSCs: Tie2 System
- Tie2-system provides information on the process determining how long it takes stem cells to reach equilibrium, and finding out how long it takes for a stem cell to complete its differentiation activity through its progenitors.
- HSC input in steady-state hematopoiesis is mostly from progenitor cells, which is different during the fetal and early postnatal life.
In Vivo Lineage Tracing of HSCs: Pdzk1ip1 system
- Method for tracing lineages and understanding how certain cells are genetically characterized
- HSCs play an important role in maintaining blood cell production
- Highlights a specific subset of cells in the continuous contribution of hematopoiesis within adult bone marrow.
In Vivo Output of HSCs
- HSCs actively contribute to maintaining steady-state hematopoiesis, supported by factors like Fgd5-CreER.
- Method for tracing lineage contributions of HSCs
- Human WASP cells are involved in gene therapy.
- Discrepancies in findings may be due to the effectiveness of labeling different systems.
Caveats
- Tamoxifen induction method affects HSC function and specificity.
What Happens at the Single Cell Level at the Steady State?
Single Cell Transplantation
- Isolating single HSCs for transplantation enables studying individual cell behavior in a way that wasn't possible before.
- This improves feasibility for single-cell transplantation experiments.
Some HSCs are better than others
- Analysis measuring the percentage of repopulating cell in specific types of transplants.
- Differences in lineage output (various cell types) are shown from different HSCs across transplants.
- Using phenotypic profiling of HSC to understand the difference in their functionality.
HSCs are heterogeneous in transplantation
- Single-cell differences in HSCs' lineage output.
- Lineage output variance and myeloid/lymphoid bias in transplantation.
What Happens at the Steady State?
- Understanding the dynamic behaviors of HSCs when they are not being stressed or stimulated from experiments.
Finding an HSC-Specific Gene
- Identifying genes specifically expressed in HSCs to differentiate them from other progenitor cells or blood cell types
Technological Advance: Next-Generation Sequencing
- Next-generation sequencing (NGS) is a powerful technology for studying gene expression (the presence and level of activity of genes within a given sample.) and other types of molecular profiles across individuals and populations.
Lineage Tracing: Lentiviral Barcoding
- Introduction of unique barcodes into stem/progenitor cells allows following the lineage development (by analyzing individual clones.)
- Reduced animal usage in experiments because the analysis is performed based on pooled cells.
- Tracking individual clones and their differentiation processes.
What is a CMP?
- Common myeloid progenitor or CMP are cells that differentiate into myeloid lineages and are heterogeneous.
Lentiviral Barcoding: Rethinking Hematopoietic Hierarchy
- Heterogeneity in CMP population.
- Lineage committed/restricted cells within the CMP population.
- Observation of cells that have myeloid/erythroid potentials within the MPP (multipotent progenitor)
- Percentage of Myeloid cells from Myeloid restricted cells.
Caveats
- Manipulation of donor cells (transduction, culture, etc.) potentially influencing experiments
- Handling the animals (irradiation) may impact results
- Interpretation of data if a single cell receives more barcodes or more than one pattern per cell.
What Happens at the Single Cell Level at the Steady State?
Technological Advance: Next-Generation Sequencing
- Comprehensive study of cells helps scientists understand the various molecular characteristics of HSCs.
- Including DNA methylation, genome accessibility, and histone modifications through various techniques.
In Vivo Lineage Tracing + scRNA-Seq: Cell Identity by Clusters
- In vivo analyses and single-cell RNA sequencing to understand how different aspects of HSC contribute to differentiation.
In Vivo Lineage Tracing + scRNA-Seq: Kinetics of Hematopoietic Differentiation
- In vivo studies and single-cell RNA sequencing to understand the differentiation process of HSCs through different developmental stages.
Conclusions
- HSC heterogeneity persists in various contexts (including population, origin, or phenotype).
- Differentiation is a continuum.
- Heterogeneity exists, even within classically defined cell populations.
- The potential for various development pathways exists for different lineages
What Happens to HSCs Over Time?
- Studies examining the impact of aging and other external factors on HSCs' functions.
HSC Functional Heterogeneity and the Impact of Aging
- Aging causes changes in HSC functionality.
- A portion of HSC cells may lose the characteristics that allow them to branch off into lymphoid lineages, skewing the balance towards myeloid development.
Clonal Hematopoiesis (CH)
- A process where a single cell acquires a mutation during hematopoiesis, leading to a clone of cells with that mutation that grow, and that can grow to a significant percentage of the blood.
What Processes Promote Clonal Hematopoiesis?
- Factors that promote CH (e.g., age, autoimmunity, and environmental exposures).
- Significance of various factors that may either initiate or drive the development of specific types of cancer or predisposition towards specific cancers.
Defining Clonal Hematopoiesis
- Various genes involved.
- DNA methylation and demethylation, and their influence on cellular development
Beyond Hematopoietic System
- Implications of CH beyond the blood system.
- Influences on cardiovascular disease, inflammation, and the tumor microenvironment.
Age-Related Defects in Hematopoiesis
- Aging impairs adaptive immune responses.
- Shifts in myeloid:lymphoid ratio.
- Increasing risk of myeloid malignancies in older individuals.
Chronic Myeloid Leukemia (CML)
- A specific type of blood cancer with observable effects based on a specific mutation.
- Common in older adults.
- Distinct disease phases (chronic, accelerated, and blast crises).
- Specific genetic lesion identified
- Treatment focuses on specific tyrosine kinase inhibitors(TKIs) and therapy strategies
What Drives the Development of CML?
- The driver for CML is the specific genetic lesion (BCR-ABL1 fusion protein).
- This mutation leads to constitutively active tyrosine kinase causing uncontrolled cell proliferation and perpetuation of CML.
Chromosomes and Cancer
- Critical research discoveries linking genetic mutations and cancer.
- The Philadelphia chromosome and its significance in CML.
BCR-ABL1 Fusion Protein
- Diagnostic marker for CML.
- The BCR-ABL1 fusion protein has an active kinase that drives CML.
- This is now a target for several treatments
BCR-ABL1 Signaling Conifers Selective Advantage
- BCR-ABL1 signaling effect on cell proliferation, survival, and cell differentiation.
Treatment of CML
- Development of tyrosine kinase inhibitors (TKIs) for CML
- Treatment strategies target BCR-ABL1.
5 Year Survival with Imatinib
- Improvement in survival rates with imatinib (a specific tyrosine kinase inhibitor)
Typical Outcomes for Chronic Phase CML
- Progression outcomes for chronic-phase CML patients following a 5 year timeframe for treatment with imatinib.
What Underlies CML Persistence/Progression?
- Factors inducing persistence or progression despite treatment.
Leukemic Stem Cells
- Specific, genetically manipulated cells that cause the perpetuation of certain cancers.
- Cells exhibiting similar characteristics to HSCs, possibly stemming from the same origin.
LSC and HSC Similarities
- Similarity in stem cell characteristics
- Potential common origin of leukemic stem cells (LSCs) based on characteristics that are similar to HSCs.
- Possible influence from BCR-ABL1 to sustain persistence, despite other therapeutic strategies.
BCR-ABL1 Independence Enables LSCs to Persist
- Development of resistance mechanisms to TKIs within LSCs.
- Mutations in BCR-ABL that lead to kinase inhibitor resistance.
Validation of HSC Origin of LSCs: In Vivo Models
- Experimental validation involving both basic and experimental transplantations involving HSCs, LSCs, and other blood cell types to confirm origin.
Perspectives for Treating CML: LSC Persistence and Blast Crisis Progression
- Strategies for treating and addressing the persistence of LSCs
- Treatments and methods to combat relapse in CML patients.
Acute Myeloid Leukemia (AML)
- Rare type of blood cancer (compared with CML).
- Significant variations in its biological behavior
- Aggressive disease progression and association with poor prognosis.
- Heterogeneity and associated poor overall survival.
AML is Associated with Poor Overall Survival
- Treatment strategies are ineffective or have limited impact on outcomes in AML patients.
It's a Little More Complicated Than BCR-ABL1
- Diverse molecular and chromosomal abnormalities associated with AML.
- Approximately 50% of AML patients have a normal karyotype (chromosome arrangement).
Mutational Burden of Cancer: Driver vs. Passenger Mutations
- Distinction between driver mutations (conferring a growth advantage) and passenger mutations (having no influence on growth.)
Identification of Additional Classes of Recurrent Mutations in AML
- Specific classes of mutations affecting different pathways identified via recurrent or frequent analysis.
Are There LSCs in AML?
- Presence of leukemic stem cells in acute myeloid leukemia (AML) confirmed by their ability to induce leukemia phenotypes in mice.
- AML, unlike CML, is more diverse.
Profile of LSCs in AML
- Gene expression profiles help in the identification of LSCs.
Currently
- Methods and strategies to identify and target LSCs in AML.
- Treatment approaches that can target specific molecules expressed on LSCs, increasing therapy effectiveness in patients.
Conclusions
- Cancer stem cells (CSCs) have a complex nature
- Defining traits of CSCs, based on genetics, phenotype, and other aspects
Characterization of Molecular Events During Early HSC Differentiation
- Understanding differentiation using methods involving sorting and single-cell RNA sequencing.
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Description
This quiz explores key concepts related to transplantation assays, focusing on the identification and tracking of donor stem cells in recipient organisms. It covers techniques such as flow cytometry and the characteristics necessary for confirming stem cell identity. Ideal for students and researchers interested in stem cell biology and regenerative medicine.
