Developmental and Stem Cell Biology 10

~25 trillion blood cells circulating in a body

2 million new blood cells per second

A multipotent stem cell at the center of blood development

Mature blood and immune cells are specialized to perform multiple tasks within the mammalian body, including providing oxygen supply and facilitating wound healing and protection from pathogens

Lethal irradiation leads to complete hematopoietic failure and eventual death of the animal.

By bone marrow transplantation after lethal irradiation

• Primary cells from certain tissues can be grown at clonal density in 2D or 3D.
• After a number of days, colonies become visible and can be assessed.
• Parameters assessed:
  • Number of colonies per input cells
  • Size of colonies
  • Differentiation s
• stem cells would give rise to bigger, less differentiated colonies, or bigger colonies with more types of cells.

+ Relatively simple and fast (compared to other assays esp. in vivo ones). Can take place immediately after tissue dissociation.

- It can be hard to obtain colonies from stem cells (eg CFU assay works better with progenitors than stem cells, this is why the LTC-IC assay was developed, but this takes a long time and risks contamination).

The now-standard mouse transplantation assay involves injection of the donor cells of interest into irradiated recipient mice (different genotype), which lack endogenous haematopoiesis. Only functionally multipotent HSCs are able to reconstitute the entire haematopoietic system within these irradiated recipients and support long-term recipient survival. However, owing to the slow reconstitution kinetics of HSCs, competitor (also termed ‘helper’) bone marrow cells are usually transplanted along with the donor cells to secure the immediate survival of the recipient by supplying HPCs, which are capable of only transiently reconstituting haematopoiesis.

• Isolation of cells from the Bone marrow
• Stain with an fluorescent antibody cocktail that recognizes specific surface markers found predominantly on HSCs

No 100% purity of functional HSC population possible! 50% is achievable.

• Remarkable heterogeneity in this population!

Limiting dilution analysis can be used to estimate the frequency of HSCs within a cell population by transplantation of various doses of cells into multiple mice. The presence or absence of one or more HSCs within the donor population is determined by assessing long-term peripheral blood reconstitution and by limiting dilution analysis performed on the basis of the number of positive versus negative mice as a function of the donor cell dose. Various reconstitution thresholds have been used in the field, but the threshold is typically set at 1% donor peripheral blood chimerism. On the basis of Poisson statistics, the number of cells (x) that result in 37% negative mice is equal to the reciprocal of the HSC frequency.

HSC self-renewal can be tracked using barcoding (or other genetic labelling) technologies. Barcoding can be performed ex vivo, by isolating HSCs from the bone marrow and labelling them in vitro with genetic barcodes (which can be applied using various methods, including transduction with a lentiviral library) and transplanted into recipient mice. Barcoding can also be performed directly in vivo, usually via the recombinase-induced or transposon-mediated ‘shuffling’ of genetic sequences to generate unique genomic sequences in each HSC. The output of individual HSCs can be determined by quantifying the barcode abundance within peripheral blood and/or bone marrow cell populations. In vivo lineage tracing technologies can be used to track HSC self-renewal in the native bone marrow without the requirement for transplantation

• fluorescent reporter expression (limited due to numbers of available fluorophores)
• Genetically encoded for example through viral transduction, transposase integration or CRISPRCas9 BC strategies 

Lineage tracing experiments aim to highlight the full progeny of a given cell/cell population through genetic tagging in situ.

--> determine the hierarchy within the hematopoietic tree!

• GFP and other fluorescent probes
• beta-galactosidase

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

+ These experiments follow lineage specification in physiological conditions

- However, they rely on complex genetic modifications and marking strategies and depend on the availability of tissue/cell population-specific promoters to drivethe expression of the recombinase.

1. Isolation of a progenitor population
2. Transplantation into lethally irradiated mice
3. Tracking of progeny in the peripheral blood

• Confetti:Random expression of one of four fluorescent proteins
• Brainbow:Random expression of multiple combinations of fluorescent proteins

+ Lineage tracing of multiple individual cells

- Permanent labelling of one cell and ALL its progenitors, no fate distinction possible afterwards

• Multiple cassettes of Cas9 barcodes that can be edited. E.g. integrate multiple Cas9 target sites all targeted by the same gRNA. The editing creates a new barcode. Can read out how many editing there are thereby follow the lineage tree that you can build by accumulating mutations

• Cas9 is highly efficient and all targets are probably hit during first few cell divisions. The Cas9 sites are destroyed very fast. No multiple editing, no long time resolution

• gRNA targeting sequence.
• Different sequences introduced in that site and followed over time.
• Site is not destroyed. Multiple sequences can be inserted

Short term ST-HSC: can rescue hematopoeisis for about 4 weeks, then depletion => Stem cell exhaustion!

Long term LT-HSC: can rescue hematopoeisiseven after secondary transplant, rare!

Most stringent: one single transplanted LT-HSC can reconstitute all cell lineages after lethal irradiation!

advantage of HUe (similar to confetti) model is that it can measure and characterize the behavior of endogenous HSC in vivo, then selectively isolate live HSCs based on fluorescent tagging, transplant them into new hosts, and study their long-term behavior in competition or under varying stress conditions.

--> showed that transplanting equal aliquots of randomly fluorescent-tagged donor HSCs into recipients resulted in an unanticipated consistency of clonal behavior in recipients in terms of cell proliferation and lineage commitment.

• mouse strain in which the fluorescent tags were driven by a ubiquitously expressed promoter with intervening stop sequences flanked by LoxP sites followed by a fluorescent cassette containing different fluorophores intercalated by multiple LoxP pairs 
• crossed with various promoter-driven Cres 
• enables Cre-induced stochastic recombination and expression.
• creates a wide range of possible colors generated by random combinations

• Each transplanted HSC retains their potential to contribute to a certain subset of lineages
• Individual HSCs exhibit a wide range of potentials upon transplantation, differing in lineage contributions and reconstitution kinetics of individual lineages, with two HSCs rarely showing identical patterns
• Immunophenotypically, these HSCs are identical! But show slight differences in transcription and potentially epigenetics that primes them for one or the other lineages

• Not distinct populations, but a continuum of different cell states.
• Each immunophenotypic defined state spans across a wide variety of transcriptional states
• Strict separation in different cell types is only visible at the extreme end of differentiation pathways
• During differentiation, cells are changing their identity not in a switch like fashion but one of multiple cell states is taken on over time

Traditional: hierarchical model, with clear distinctions between the Stem cell state and all the progenitors

Revised model: HSCs are heterogenous and are skewed towards one or multiple lineages, this skew is present in the HSC state and established early in development but unclear when and how

1. Heterogeneity in gene expression
2. Epigenetic modifications
3. biological processes
4. segregation of cell fate determinants
5. stochasticity
6. niche localization

across all the different populations including HSCs  (Just based on surface marker expression, there is a strong heterogeneity eithin the sorted population)

mostly DNA methylation that might restrict potential of HSC to enter one or another cell state (HSC with different potential show stable differences in DNA methylation patterns)

• HSC with loss of Dnmt3a can be transplanted indefinitely, gaining essentially immortality
• Outcompete WT cells in competition assays
• not able to rescue it again by reestablishing DNA methylation

Many biological processes are coupled: cell size increases during cell cycle, therefor metabolic activity and transcriptional activity overall increase and could affect cellular differentiation behaviour

Different microenvironment within the bone marrow niche that could influence HSC behaviour

The stem cell niche is the in vivo microenvironment where stem cells both reside and receive stimuli that determine their fate. Therefore, the niche should not be considered simply a physical location for stem cells, rather as the place where extrinsic signals interact and integrate to influence stem cell behaviour.

In humans: axial skeleton such as cranium sternum, ribs, vertebrae and ilium are the major source of hematopoiesis HSCs isolated from different bones in the body have similar gene expression profiles and capabilities

1. Adult stem cells differentiate when out of their natural environment.
2. During development, daughter cells in different environments acquire different fates.

• The niche can exist even in the absence of stem cells.
• Niche cells can be absent and replaced by ECM.
• Cell-cell and cell-matrix contacts are essential.
• Blood vessels are always present in the vicinity.
• Neuronal inputs have increasingly been described.

Cxcl12 and SCF (aka KIT ligand) as main growth factors that are produced by multiple cells within the bone marrow, including mesenchymal stem cells (MSCs) that are found perivascular

MSC and their progenitors are potentially the main organizers of the HSC niche

conditional KO with Cre

Cre expression: tissue specific through using a cell type specific driver, for example NG2

For a better control: Cre-ERT2 fusion => Nuclear localization domain from the estrogen receptor that reacts specifically to the presence of tamoxifen. When tamoxifen is present (for example through the drinking water) Cre enters the nucleus and recombines the two Lox sites

+ Allows for the analysis for cell type specific gene effects

- Cre driver are often not as specific as wanted (example Nes cre) or are expressed in other cell types and might have pleiotropic effects

• Intravital imaging
• New clearing technologies
• High resolution imaging technologies plus sophisticated image analysis software

• HSC are distributed throughout the bone marrow, often close to the vasculature. But there is no preference for a specific kind of vasculature!
• it cannot be excluded that different HSCs in different locations can be activated differently! Different oxygen levels could lead to different ROS levels could lead to different activation kinetics!

Ongoing research, Current advances are substantial, but still not able to fully preserve HSC capabilities.

As the HSC population can expand substantially in its native niche, an understanding of the mechanisms of HSC maintenance is a prerequisite for the development of protocols to successfully expand HSC populations ex vivo for transplantation