Hematopoietic Stem Cell Development Is Dependent on Blood Flow

Hematopoietic Stem Cell Development Is Dependent on Blood Flow. Trista E. North, Wolfram Goessling, Marian Peeters, Pulin Li, Craig Ceol, Allegra M. Lord, Gerhard J. Weber, James Harris, Claire C. Cutting, Paul Huang, Elaine Dzierzak, and Leonard I. Zon. Cell. May 2009; (137)4: 736-748.

Available at: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4W9297H-K&_user=4420&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=5be731494083d83e4456767fea553c35

It is important to look at the development of hematopoietic embryonic stem cells because the regulation of development is very similar to that of the maintenance and recovery for adult hematopoietic stem cells (HSC). As such, learning the development of HSCs can give insight into potential treatments for aging and damaged tissue. One particularly interesting location of study is the aorta-gonads-mesonephros (AGM) region of a heart during embryogenesis due to the fact that this region is densely seeded with hematopoietic stem cells. This paper uses the AGM region as a model to test the hypothesis that mechano-transduction from pulsatile blood flow helps HSCs develop during embryogenesis. In addition this paper attempts to elucidate key pathway intermediates in order to identify which pathways are being affected by mechano-transduction in order to promote HSC development.

To begin with, this paper attempts to establish that HSC development is enhanced by pulsatile blood flow. The reason for such suspicion is that during early development, the hearts of vertebrates starts to pump even though they can easily rely on diffusion for oxygenation. One possible explanation is the heart starts to pump in order to promote HSC cell development in the AGM. In order to test this theory, the researchers used a mutant zebra fish called silent heart (sih) in which the heart does not beat during embryogenesis. In the absence of blood flow during embryogenesis, there is less development. Development was measured in number of HSCs present, inferring that a higher number means superior development.

Initially, the researchers tried to determine what factors/ signaling pathways were at play. First they wanted to confirm that regulators can affect development. Drawing from knowledge from literature, they postulated that factors runx1/cmyb+ are directly related to HSC development. They tried various inhibitors and enhancers to determine what key intermediates are affecting the runx1/cmyb+ concentration. After trying a menagerie of inhibitors and enhancers, they elucidated a lot of effectors such as: digoxine, L-NAME, etc. Looking at aorta diameter, HSC count and runx1/cmyb+ levels, they concluded there are many factors that can contribute to development including nitric oxide (NO).

Next, they inferred that if in fact mechano-transduction is lowering some regulatory pathway in order to slow down HSCs growth, then one can “rescue” these cells with no mechano-transduction by fixing the regulation. The researchers determined that it was NO that was the major contributor in HSC development because NO was able to rescue zebra fish development in the absence of pulsatile flow. They also reconfirmed this by knocking out the downstream pathway for NO signaling and confirming that this has the same effect as knocking out NO.

Next, the researchers established that NO signaling affects development prior to mechano-transduction. They tested this by adding in a NO inhibitor (L-NAME) and seeing impeded growth. The researchers also wanted to see if the effect of NO signaling was cell autonomous. They achieved this by transplanting NO treated cells into a blastula and watching only the transplanted cells develop. They distinguished transplanted cells from their neighbors by a fluorescent marker. As well, they were interested in other pathways talking with NO signaling, namely the wnt and notch pathways. They used a mindbomb mutant (mb) in which the notch pathways was greatly knocked down. This showed a greatly reduced hematopoietic stem cell development. They also had a constitutively on notch mutant in which reduced HSCs showed increase development and they were able to knock down this development by adding a NO inhibitor, L-NAME. They confirm from this that notch is upstream of NO. The same scheme was used on the wnt pathway.

For completeness, they attempted to look at other pathways developed by blood flow. In order to search for these pathways, they look for phylogenetic trees and genomic similarities in order to determine similar pathway intermediates. They found nos1 and nos3, both NO synthases in other cell types. Reduction of NO resulted in reduced development in those cell types.

Lastly, they confirmed that what they saw in zebra fish is conserved in mice, by taking using the L-NAME inhibitor and using it in pregnant mice and doing histological studies to determine reduced development of hematopoietic stem cells.

Discussion and Criticism

One improvement (nice way of calling something wrong) to this paper is that in the cell autonomy part of the paper. The paper claims that the effect of nos1 (which is supposed to reduce HSC growth) is not context dependent (not affected by neighboring cells.) To show this is autonomous of the context the cells are in, they took nos1 knockdown mutants and implanted that into a blastula of healthy cells. Tracking of these cells was done with a fluorescent GFP tag. The one criticism to this is that while the experiment does suggest that this is context dependent it is not completely conclusive. The problem is that you are transplanting an ensemble of nos1 knockdowns into the blastula. Unfortunately, by transplanting a group of cells, you may be transplanting an environment. It is possible that the small isolated colony of cells may regulate themselves such that they do not develop. What I would do to be more conclusive is that I would want to transplant a single cell into the new environment to check for cell autonomy.

Another potential improvement is in the interacting signaling pathways. It is very difficult to assess the behavior of a system of pathways from a simple trial and error test. The paper identifies that both notch and wnt pathways positively affects HSCs by increasing NOS. However, this assumes that the relationships are linear. This is probably not the case as it can be very possible that wnt and notch both, by themselves, increases development, but together, they it will reduce HSC development. Furthermore, it is suggested that increased NO is the only factor. This might in fact be an ensemble effect. To improve this into more meaning and predictive information, I would construct consult a systems biologist in order to construct a web or pathway instead of single interaction. Then I would test interacts based on possible topologies to confirm and conclude a model of signal transduction.

As far as big picture problems go, I would do more tests to confirm the relevance of these findings in developmental systems in mature systems. The end goal of these experiments is to help with tissue repair in adult systems. As such, this information would only be useful if it does in fact pertain to adult systems. As such, I would run experiments in mature cells to see if I can use NO signaling or flow to help wound healing or tissue regeneration.

Furthermore, due to the final goal of human application, I would also try to test to see if I see the same conclusions in human cells. Mouse and fish models are not always analogous to humans. One example of this would be the drug for morning sickness: thalidomide in which there was no negative effect in mice, but it caused human children to have missing limbs. To account for this, I would confirm my results in primates which are much closer to humans.

Lateness excused due to iGem