Molecular and functional heterogeneity in the human haematopoietic stem cell compartment

Speaker: Elisa Laurenti
Department: Cambridge Stem Cell Institute, University of Cambridge, Cambridge
Location: Erasmuc MC Rotterdam
Date: Juni 12, 2017
Author: Teun Huijben

Elisa Laurenti has been interested in stem cells during her entire academic carrier. After doing a PhD and PostDoc in this field, she now has her own lab at Cambridge, where she studies the heterogeneity in the human haematopoietic stem cell (HSC) compartment. In this one hour she introduced us to the field and explained the research she has performed in the last years.

The main point of Elisa’s talk was that where we all think of stem cells as just stem cells, there is actually a large heterogeneity between them. By quantifying the differences between the different HCSs, she hopes to define distinct subsets with different functions, characteristics and detectable markers within the broad HSC-pool.

By single-cell analysis, Elisa found two distinct subsets of haematopoietic stem cells: long-term HSCs (LT-HSC) and short-term HSCs (ST-HSCs). They are characterized by just two surface markers: LT-HSCs express high levels of CDf49 and low levels of CD90, where ST-HSCs have opposite expression levels. The functional difference between them is that LT-HSCs divide very rarely, and ST-HSCs divide more often.

Transcriptional analysis of LT-HSCs and ST-HSCs didn’t give any results, they both showed the same expression landscapes. One explanation for this could be that both cells are very quiescent and therefore not transcriptionally active. The solution Elisa and her colleagues found was to activate the cells and then analyse their transcriptomes. Once activated, the cells start in quiescence, which is a ’sleeping’ state, and are then activated. The ST-HSCs are activated earlier than LT-HSCs, which is another functional difference between them.

To activate the in vitro cultured HSCs, they are transplanted into living mice or into in vitro cultured tissues. Both activated cells are analyzed by single RNA-seq and microarrays. By doing this, they found at least 34 genes that are differently expressed between two subsets. CDK6 appeared to have to most distinct difference in expression between the two groups and was the best gene to indicate whether a cell is ST-HSC or LT-HSC. Surprisingly, treatment with CDK6 determined the state of the cells: over-activation of CDK6 resulted in a faster activation and a CDK6 inhibitor resulted in slower activation.

However, next to this direct effect by changing the expression level of CDK6, also long-term effects were measured. When CDK6 was over-expressed, LT-HSCs gained a positive competitive advantage over SC-HSCs over the long term. In other words, they outnumbered the ST-HSCs. This can be explained by the fact that CDK6 stimulates activation of the cells. ST-HSCs already activate quite fast, so stimulating activation results in activation of all ST-HSC. They all start differentiating and no ST-HSC will be left. LT-HSCs on the other hand, activate more slowly and will remain abundant in the HSC-pool, and will eventually dominate over the ST-HSCs in number.

In the remainder of the time, Elisa told about her current research in further defining subsets of haematopoietic stem cells by finding new markers that characterize distinct groups. Her talk emphasized once more the difficulties we face when looking at stem cells, or molecular biology in general; tissues are very heterogenous and we do not yet know a lot about all their differences. However, her talk was very clear and she is obvious an important person in this field.

When DNA replication runs into a problem

Hematology Seminar by Puck Knipscheer

Erasmus Medical Center, Department of Hematology 19-1-2015

At the moment, Puck Knipscheer is the group leader of the Knipscheer research group at the Hubrechts Institute Utrecht. This group is currently trying to get a better understanding of the so called Fanconi Anemia pathway (FA pathway).  This pathway plays an important role in the DNA repair mechanism that repairs interstrand crosslinkings (ICLs). As can be seen in figure 1, interstrand crosslinking (ICL) is the phenomenon that the two strands of DNA are linked together and can be induced by exo- and endogenous agents such as carmustine and nitrogen mustard, which are agents used in chemotherapy. This means that understanding interstrand crosslinking and its repair mechanisms could be and already is very useful in treatment of cancer.

puckhematology
Your body, and that of many other animals, has a repair mechanism to repair interstrand cross links and as said, the FA pathway plays an important role in this. What the FA pathway basically does is coordinating and initiating several DNA repair mechanisms. This is done by the ubiquitination of the proteins FANCI and FANCD2, who in their turn activate other repair mechanisms like homologous recombination, nucleotide excision repair and mutagenic translesion synthesis. People who have a mutation in their DNA that prevents the pathway from being executed suffer from the genetic disease Fanconi Anemia (FA). These people have several symptoms, like developmental defects, bone marrow failure, increased cancer risks and cellular sensitivity to ICLs. Understanding the pathway, like Puck Knipscheer aims to do, can therefore contribute to both cancer treatments as well as understanding and perhaps healing of FA. DNA replication and repair is studied Xenopus Laevis (African clawed frog) egg extracts, because they are known to have unique form eukaryotic DNA replication and are fully soluble.

Kasper Spoelstra – wkspoelstra@hotmail.nl