SC assays

Question 1

What is a colony formation assay?

Answer 1

You grow cells at clonal density and see if you can grow anything. Colonies with stem cells present are expected to grow the most

Question 2

What cells are used for colony formation assays?

Answer 2

probably primary cells

Question 3

What parameters are assesed in colony formation assays?

Answer 3

• parameters assesed:
  
  • number of colonies per input cells
  • size of colonies
  • difeferentiation

Question 4

What is the benefit of colony formation assays?

Answer 4

relatively simple and fast . Can take place immediately after tissue dissociation

Question 5

What can be a downside of colony formation assays?

Answer 5

it can be hard to obtain colonies from stem cells

Question 6

What is a potential usecase for a colony formation assay?

Answer 6

A clonogenic assay is the most direct quantitative method of measuring human hematopoietic progenitor cellsin vitro Hematopoietic colonies are essentially clones of cells produced by a single progenitor cell. The aim of colony-forming unit (CFU) assays is to define the potential of hematopoietic stem and progenitor cell populations for proliferation and lineage differentiation.

Question 7

What is a sphere formation assay?

Answer 7

primary cells from certain tissues can be grown in the absence of feeder cells in three dimensional sphere culture

Question 8

What can you use sphere formation assays for?

Answer 8

• it is believed that only stem cells cam give rise to serial spheres
• sphere formation is an assay that can be performed to obtain an indication of the presence of stem cells in a poorly characterised tissue

Question 9

What might be the downside of sphere formation assays?

Answer 9

it is impossible to avoid differentiation within the sphere and to control/study molecular signals exchanged between cells within the spheres

Question 10

What are organoids?

Answer 10

Organoids are tiny, self-organized three-dimensional tissue cultures that are derived from stem cells. Such cultures can be crafted to replicate much of the complexity of an organ, or to express selected aspects of it like producing only certain types of cells.

Question 11

Why can organoids be particularly appealing?

Answer 11

Most of what we know about embryonic development has been learned by extrapolating to human biology what is observed in mice and other animal models. Now, thanks to organoids, researchers have the possibility of culturing tiny versions of each tissue using human cells.

Question 12

What does Carla Kim study and what can it be helpful with?

Answer 12

Researchers like Carla Kim, PhD are using organoids to determine the role of stem cells in tissue regeneration, maintenance, and function and to understand how these cells talk to each other. Kim and her group were the first scientists to grow lung organoids that mimic two distinct parts of the lung: the airways and the alveolar sacs where gas exchange occurs. They did it by using a special culture setup that allowed the cells to be in contact with both air and liquid, mimicking the lung environment. Their culture also included helper cells derived from blood vessels to stimulate stem cell growth.

Question 13

Why would you induce in vitro differentiation assays on stem progenitor cells?

Answer 13

• for some stem progenitor cells it is possible to induce differentiation in vitro in order to:
  
  • evaluate the differentiation potential of distinct cell populations
  • evaluate the role of genes/compounds in regulating differentiation along a specific lineage

Question 14

What do in vitro differentiation assays allow?

Answer 14

• allow retrospective identification of stem/progenitor cells (you mnake them do something and then you can tell what cell it actually was)
• used to see what the tissue you have can actually do and then you can take it to further experiments
• allow detailed analysis of proliferation and differentiation potential

Question 15

What are some benefits and issues with in vitro differentiation assays?

Answer 15

• very highly controlled
• do not necessarily reflect what happens in vivo

Question 16

What does transplantation tell you?

Answer 16

assays give an indication of the proliferative and multi-lineage potential of the cell studied

Question 17

How are transplanted cells tracked?

Answer 17

• transplanted cells and their progeny are marked so they can be recognised
• common markers: fluorescent proteins, Y chromosome

Question 17

What are some disadvantages of transplantation?

Answer 17

• unfortunately they can be laborious and time consuming
• test stem cells in conditions very far from physiological

Question 18

What is lineage tracing used for?

Answer 18

lineage tracing experiments tend to highlight the full progeny of a given cell/cell population through genetic tagging in situ

Question 19

What are some commonly used tags for lineage tracing?

Answer 19

• commonly use tags:
  
  • GFP and other fluorescent probes
  • beta-galactosidase

Question 20

What is the principle of genetic lineage tracing?

Answer 20

genetic lineage tracing takes advantage of recombinases expressed in specific tissues to recognise and excise specific DNA sequences, leading to genetic tagging and reporter expression in the entire progeny of the cells expressing the recombinase

Question 21

How does Cre work in lineage tracing?

Answer 21

Cre cuts the DNA and pulls it back together . When Cre is expressed in transgenic mice it cuts out the lox parts and the STOP part is lost leading to expression of the GFP

Question 22

What is the advantage of using CreER over regular Cre?

Answer 22

CreER (estrogen receptor) has become very popular as it allows us to keep the activity of Cre but it is only activated when add tamoxifen as ensured by the ER part

Question 23

Whatt are some advantages and limitations of lineage tracing experiments?

Answer 23

• experiments follow lineage specification in physiological conditions
• unfortunately they rely on complex genetic modifications and marking strategies and depend on availability of tissue specific promoters to drive the expression of the recombinase
• if you label progenitors instead of stem cells at some point your progenitros will die and your tracing will be lost

Question 24

What is the concept of brainbow?

Answer 24

"Brainbow" mice are engineered with a gene that includes three different fluorescent proteins, but only one color is actually expressed from each copy of the DNA construct. Pairs of "incompatible lox sites" are nested around different portions of the gene, allowing for recombination to snip out different parts of the gene randomly. Depending on what DNA is excised, a different color results.

Question 25

What is the concept of confetti?

Answer 25

A variation of the "Brainbow" gene adds a transcriptional "roadblock" to inhibit expression of the fluorescent proteins until Cre is activated. By placing Cre under the control of a tissue-specific promoter, the "Brainbow" effect can be activated in one tissue of the mice. This is called the "Confetti" mouse.

Question 26

Explain competition in the crypt in the concept of confetti

Answer 26

Here is an image of the small intestine, zoomed in on the crypts. The "Confetti" gene was activated in all cells in the intestine, including stem cells. Once the Cre recombination process is over, each stem cell produces progeny that inherit the same fluorescent marking. The number of cells in the crypt is fixed. Thus, over time, stem cells (~14) compete each other out of this niche, and the crypt becomes clonal, visualized here by monochromatic crypts.

Question 27

What is barcoding?

Answer 27

- it is based on carrying specific DNA sequences - you can induce expression of a semi random sequence in the stem cell and because of the way it is generated each stem cell will have a unique barcode and then you can see which progeny carries the same barcode

Question 28

What advantage does barcoding have over brainbow?

Answer 28

it works similar to brainbow but is easier to read as you get it straight from sequencing. By tagging bone marrow cells of mice with a genetic label, or barcode, researchers were able to track and describe the family tree of individual blood cells as they form in their natural environment

Question 29

What is multiplex immunofluorescence?

Answer 29

multiplex immunofluorescence -Conventional immunohistochemistry (IHC) is commonly used as a diagnostic technique in the field of tissue pathology but suffers from certain limitations. The most critical of these is that this technique only permits the labelling of a single marker per tissue section. This results in missed opportunities to gain important prognostic and diagnostic information from patient samples. Multiplex Immunohistochemistry/Immunofluorescence (mIHC/IF) technologies, which allow the simultaneous detection of multiple markers on a single tissue section, have been introduced and adopted in both research and clinical settings in response to increased demand for improved techniques. Such techniques provide a comprehensive view of marker distribution and tissue composition, and are poised to solve major questions surrounding the pathogenesis of various complex disorders. The ability to label multiple markers on a single section is of particular significance when studying samples taken from rare donors, where tissues may be of low availability.

Question 30

What is spatial trascriptomics?

Answer 30

spatial transcriptomics - is a method for assigning cell types (identified by the mRNA readouts) to their locations in the histological sections. Defining the spatial distribution of mRNA molecules allows for the experimentalist to uncover cellular heterogeneity in tissues, tumours, immune cells as well as determine the subcellular distribution of transcripts in various conditions.This information provides a unique opportunity to decipher both the cellular and subcellular architecture in both tissues and individual cells.

Question 31

What is spatial proteomics?

Answer 31

spatial proteomics -Spatial Proteomics is study of spatial organization of proteins within cells. High-throughput imaging to visualize all cellular proteins and quantitative mass spectrometry are two widely used approached to spatial proteomics.