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Editor | The differentiation pathways of stem cells and precursor cells are different at the single cell level, and individual stem cells have different preferences in the process of differentiating into different mature cells.
Experiments have shown that this fate preference is not completely random or only affected by extracellular signals, and is also related to the state of the cell [1].
The rapid development of single-cell sequencing technology in recent years has greatly expanded our understanding of the heterogeneity of stem cell transcriptomes.
Because single-cell sequencing is destructive, and the gene expression and subsequent differentiation of stem cells occur at different times.
Unless you turn back time, after sequencing a stem cell, its differentiation path is unknown.
Dendritic cells (DC) connect innate immunity and adaptive immunity in the body, mainly including type 1/2 conventional dendritic cells (type 1/2 conventional DC, cDC1, cDC2), and plasmacytoid dendrites Cells (plasmacytoid DC, pDC) [2].
How different types of DC are differentiated and regulated by hematopoietic stem cells (HSC) is not yet fully understood.
Is the heterogeneity of hematopoietic stem cells at the transcriptome level related to the fate preference of different types of DC? In order to solve this problem, on April 15, 2021, the Australian Shalin Naik team published Clonal multi-omics reveals Bcor as a negative regulator of emergency dendritic cell development in Immunity, by isolating and differentiated early sister cells and performing sequencing and Functional tests link the cell fate and stem cell gene expression, which occur at different times.
The study uses the in vitro differentiation system from hematopoietic stem cells to dendritic cells as an example, which correlates the transcriptome heterogeneity of hematopoietic stem cells and different DC fate preferences.
And further screening and verifying Bcor as a negative regulatory gene of cDC2 and pDC through CRISPR, and revealing the different roles that Bcor plays in inhibiting the differentiation of cDC2 and pDC.
The author first explored the ability of FLT3L-mediated hematopoietic stem cell differentiation early (day 2.
5-4.
5) of sister cells to finally differentiate into three different DCs, and found that sister cells from the same hematopoietic stem cell have a highly consistent differentiation path preference .
For example, a certain sister cell from the same hematopoietic stem cell has a strong preference for cDC2, and less differentiation into cDC1 and pDC.
The similarity of the fate preferences of sister cells allows them to be separated into multiple groups, with some cells undergoing transcriptome sequencing and the other cells continuing to undergo in vitro differentiation (SIS-seq).
After independently isolating and sequencing multiple sister cells of hematopoietic stem cells, the authors used a generalized linear model with regularization to find the relationship between stem cell gene expression and fate preference, and screened more than 400 genes.
It includes a number of genes known to regulate DC differentiation, such as Id2, Batf3, Irf4, etc.
, which proves the role of this method in discovering genes that regulate the fate of stem cells.
Not only that, the screened list also contains a large number of unreported genes.
For example, stem cells with high expression of Bcor, their sister cells will differentiate into cDC1 rather than cDC2 and pDC.
In order to further verify the regulatory functions of these genes, the authors used CRISPR to perform high-throughput screening of these genes and confirmed a number of new genes that regulate DC differentiation.
Among them, the hematopoietic stem cells that have been knocked out of Bcor will differentiate into cDC2 and pDC in large numbers, but the number of cDC1 does not increase significantly, which is consistent with previous experiments.
At the same time, the authors also found that knocking out Bcor increased the precursor cells (pre-DC) of suspected DCs.
In order to better characterize the impact of Bcor knockout on the transcriptome at the single-cell level, the authors also performed single-cell sequencing on the cells on the DC differentiation pathway after Bcor knockout and the blank control.
After data integration, the expression of key markers in mature cells cDC1, cDC2 and pDC that were knocked out of Bcor were similar.
At the same time, experiments showed that mature DC cells differentiated after knocking out Bcor can still perform their functions, indicating that Bcor may not affect mature DC.
Although the gene expression of mature cells is similar, stem cells after Bcor knockout differentiate into a large number of precursor cells.
Through comparison with the known database Immgen, it is found that the transcriptome of these precursor cells is similar to the different differentiation stages of hematopoietic stem cells (MPP, CDP, CLP).
Combining the results of multiple experiments, the stem cells after Bcor knockout will generate a large number of DC precursor cells, and these cells have the differentiation preference of cDC2 and pDC.
In in vivo experiments, the authors found that Bcor does not affect the fate choice of DC differentiation under steady-state conditions.
Only in the in vivo environment stimulated by FLT3L can the number of cDC2 and pDC increase significantly, indicating that Bcor's regulation of cDC2 and pDC is dependent For FLT3L.
Although the positive regulatory effect of Bcor knockout on cDC2 and pDC has been confirmed, at the level of a single stem cell clone, this regulation may have multiple manifestations.
For example, stem cells that originally lacked the differentiation ability of cDC2 and pDC, and whose fate was biased toward cDC1 gained the differentiation ability (fate gain) of cDC2 and pDC after knocking out Bcor, or stem cells that could originally generate three types of DCs.
Later, the differentiation pathway of cDC2 and pDC was strengthened, and the number of cells was expanded (clonal expansion). Using a similar idea, the author added DNA barcodes to hematopoietic stem cells for lineage tracing, and used sister cells from the same hematopoietic stem cells to conduct separate experiments.
Some cells were knocked out of Bcor, and the other part of the cells was used as a blank control to explore the effect of Bcor on stem cells.
What is the impact of its own cell fate decision (SIS-skew).
Experimental results show that the increase in the number of pDC cells is mainly due to fate gain, that is, cells that originally lack pDC differentiation ability can differentiate into pDC.
The increase in the number of cDC2 cells is mainly due to clonal expansion, that is, stem cells that can differentiate into cDC2 originally, and their ability to differentiate and reproduce has been strengthened.
This experiment further revealed the functional differences of Bcor in regulating the differentiation of different DCs.
In general, the article provides a general idea for studying the differentiation of stem cells to correlate the state of stem cells and their differentiation pathways.
This method is not limited to the transcriptome.
It can also be used to separate sister cells into multiple groups to perform multi-omics studies at the clonal level, or to separate them at multiple time points to study the dynamic differentiation trajectory [3].
It is also possible to use expressible DNA barcodes to simultaneously label multiple hematopoietic stem cells and isolate sister cells to increase throughput [4].
Tian Luyi, Sara Tomei, Jaring Schreuder are the co-first authors of this article.
Original link: https://doi.
org/10.
1016/j.
immuni.
2021.
03.
012 Platemaker: 11 References 1.
Naik, S.
, Perié, L.
, Swart, E.
et al.
Diverse and heritable lineage imprinting of early haematopoietic progenitors.
Nature 496, 229–232 (2013).
https://doi.
org/10.
1038/nature120132.
Shortman, K.
, Liu, YJ.
Mouse and human dendritic cell subtypes.
Nat Rev Immunol 2, 151 –161 (2002).
https://doi.
org/10.
1038/nri7463.
Lin, DS, Tian, L.
, Tomei, S.
et al.
Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development.
Nat Cell Biol 23, 219–231 (2021).
https://doi.
org/10.
1038/s41556-021-00636-74.
Weinreb C, Rodriguez-Fraticelli A, Camargo FD, Klein AM.
Lineage tracing on transcriptional landscapes links state to fate during differentiation.
Science.
2020;367(6479):eaaw3381.
doi:10.
1126/science.
Aaw3381 Reprinting Instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
Experiments have shown that this fate preference is not completely random or only affected by extracellular signals, and is also related to the state of the cell [1].
The rapid development of single-cell sequencing technology in recent years has greatly expanded our understanding of the heterogeneity of stem cell transcriptomes.
Because single-cell sequencing is destructive, and the gene expression and subsequent differentiation of stem cells occur at different times.
Unless you turn back time, after sequencing a stem cell, its differentiation path is unknown.
Dendritic cells (DC) connect innate immunity and adaptive immunity in the body, mainly including type 1/2 conventional dendritic cells (type 1/2 conventional DC, cDC1, cDC2), and plasmacytoid dendrites Cells (plasmacytoid DC, pDC) [2].
How different types of DC are differentiated and regulated by hematopoietic stem cells (HSC) is not yet fully understood.
Is the heterogeneity of hematopoietic stem cells at the transcriptome level related to the fate preference of different types of DC? In order to solve this problem, on April 15, 2021, the Australian Shalin Naik team published Clonal multi-omics reveals Bcor as a negative regulator of emergency dendritic cell development in Immunity, by isolating and differentiated early sister cells and performing sequencing and Functional tests link the cell fate and stem cell gene expression, which occur at different times.
The study uses the in vitro differentiation system from hematopoietic stem cells to dendritic cells as an example, which correlates the transcriptome heterogeneity of hematopoietic stem cells and different DC fate preferences.
And further screening and verifying Bcor as a negative regulatory gene of cDC2 and pDC through CRISPR, and revealing the different roles that Bcor plays in inhibiting the differentiation of cDC2 and pDC.
The author first explored the ability of FLT3L-mediated hematopoietic stem cell differentiation early (day 2.
5-4.
5) of sister cells to finally differentiate into three different DCs, and found that sister cells from the same hematopoietic stem cell have a highly consistent differentiation path preference .
For example, a certain sister cell from the same hematopoietic stem cell has a strong preference for cDC2, and less differentiation into cDC1 and pDC.
The similarity of the fate preferences of sister cells allows them to be separated into multiple groups, with some cells undergoing transcriptome sequencing and the other cells continuing to undergo in vitro differentiation (SIS-seq).
After independently isolating and sequencing multiple sister cells of hematopoietic stem cells, the authors used a generalized linear model with regularization to find the relationship between stem cell gene expression and fate preference, and screened more than 400 genes.
It includes a number of genes known to regulate DC differentiation, such as Id2, Batf3, Irf4, etc.
, which proves the role of this method in discovering genes that regulate the fate of stem cells.
Not only that, the screened list also contains a large number of unreported genes.
For example, stem cells with high expression of Bcor, their sister cells will differentiate into cDC1 rather than cDC2 and pDC.
In order to further verify the regulatory functions of these genes, the authors used CRISPR to perform high-throughput screening of these genes and confirmed a number of new genes that regulate DC differentiation.
Among them, the hematopoietic stem cells that have been knocked out of Bcor will differentiate into cDC2 and pDC in large numbers, but the number of cDC1 does not increase significantly, which is consistent with previous experiments.
At the same time, the authors also found that knocking out Bcor increased the precursor cells (pre-DC) of suspected DCs.
In order to better characterize the impact of Bcor knockout on the transcriptome at the single-cell level, the authors also performed single-cell sequencing on the cells on the DC differentiation pathway after Bcor knockout and the blank control.
After data integration, the expression of key markers in mature cells cDC1, cDC2 and pDC that were knocked out of Bcor were similar.
At the same time, experiments showed that mature DC cells differentiated after knocking out Bcor can still perform their functions, indicating that Bcor may not affect mature DC.
Although the gene expression of mature cells is similar, stem cells after Bcor knockout differentiate into a large number of precursor cells.
Through comparison with the known database Immgen, it is found that the transcriptome of these precursor cells is similar to the different differentiation stages of hematopoietic stem cells (MPP, CDP, CLP).
Combining the results of multiple experiments, the stem cells after Bcor knockout will generate a large number of DC precursor cells, and these cells have the differentiation preference of cDC2 and pDC.
In in vivo experiments, the authors found that Bcor does not affect the fate choice of DC differentiation under steady-state conditions.
Only in the in vivo environment stimulated by FLT3L can the number of cDC2 and pDC increase significantly, indicating that Bcor's regulation of cDC2 and pDC is dependent For FLT3L.
Although the positive regulatory effect of Bcor knockout on cDC2 and pDC has been confirmed, at the level of a single stem cell clone, this regulation may have multiple manifestations.
For example, stem cells that originally lacked the differentiation ability of cDC2 and pDC, and whose fate was biased toward cDC1 gained the differentiation ability (fate gain) of cDC2 and pDC after knocking out Bcor, or stem cells that could originally generate three types of DCs.
Later, the differentiation pathway of cDC2 and pDC was strengthened, and the number of cells was expanded (clonal expansion). Using a similar idea, the author added DNA barcodes to hematopoietic stem cells for lineage tracing, and used sister cells from the same hematopoietic stem cells to conduct separate experiments.
Some cells were knocked out of Bcor, and the other part of the cells was used as a blank control to explore the effect of Bcor on stem cells.
What is the impact of its own cell fate decision (SIS-skew).
Experimental results show that the increase in the number of pDC cells is mainly due to fate gain, that is, cells that originally lack pDC differentiation ability can differentiate into pDC.
The increase in the number of cDC2 cells is mainly due to clonal expansion, that is, stem cells that can differentiate into cDC2 originally, and their ability to differentiate and reproduce has been strengthened.
This experiment further revealed the functional differences of Bcor in regulating the differentiation of different DCs.
In general, the article provides a general idea for studying the differentiation of stem cells to correlate the state of stem cells and their differentiation pathways.
This method is not limited to the transcriptome.
It can also be used to separate sister cells into multiple groups to perform multi-omics studies at the clonal level, or to separate them at multiple time points to study the dynamic differentiation trajectory [3].
It is also possible to use expressible DNA barcodes to simultaneously label multiple hematopoietic stem cells and isolate sister cells to increase throughput [4].
Tian Luyi, Sara Tomei, Jaring Schreuder are the co-first authors of this article.
Original link: https://doi.
org/10.
1016/j.
immuni.
2021.
03.
012 Platemaker: 11 References 1.
Naik, S.
, Perié, L.
, Swart, E.
et al.
Diverse and heritable lineage imprinting of early haematopoietic progenitors.
Nature 496, 229–232 (2013).
https://doi.
org/10.
1038/nature120132.
Shortman, K.
, Liu, YJ.
Mouse and human dendritic cell subtypes.
Nat Rev Immunol 2, 151 –161 (2002).
https://doi.
org/10.
1038/nri7463.
Lin, DS, Tian, L.
, Tomei, S.
et al.
Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development.
Nat Cell Biol 23, 219–231 (2021).
https://doi.
org/10.
1038/s41556-021-00636-74.
Weinreb C, Rodriguez-Fraticelli A, Camargo FD, Klein AM.
Lineage tracing on transcriptional landscapes links state to fate during differentiation.
Science.
2020;367(6479):eaaw3381.
doi:10.
1126/science.
Aaw3381 Reprinting Instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights and offenders must be investigated.
