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.

Induced Pluripotent Stem Cells for treatment of cardiovascular and respiratory diseases

Speaker:             Ulrich Martin

Department:     Cell biology

Location:            Erasmus MC Rotterdam

Date:                    15-5-2017

Author:               Katja Slangewal

The endogenous heart regeneration after a myocardial infarct is far from sufficient in mammals. Actually, less than 50% of the cardiomyocytes in a mammal heart are replaced during the entire life span. These facts immediately show the importance of the development of stem cell based regenerative treatments. This is the main area of research for Ulrich Martin from the Hannover Medical School, centre for Regenerative Medicine. During his talk, he did not only talk about the use of induced pluripotent stem cells (iPSCs) as therapeutic for heart repair, but also about the use of patient specific iPSCs in context for cystic fibrosis (a severe monogenic disease).

It has become more and more clear that quite often a general type of disease differs a lot from patient to patient. Emphasizing the need of patient specific treatments. Martin and his team work on patient specific iPSCs for disease modelling and the search to new drugs. The production of iPSCs (figure 1) becomes more and more efficient. The reprogramming step has become routine and it is even possible to use big tanks for the production of large numbers of iPSCs. This in contrast to a few years ago, when the production of iPSCs was tedious, time consuming, intensive and about low numbers of cells. The automatization and upscaling of stem cell production however was needed for high throughput and industrialization. For the first time in history, even big pharma are interested in stem cells.

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Figure 1: iPSCs are formed by taking somatic cells (for instant adult fibroblast cells) and adding reprogramming factors (KLF4, SOX2, c-Myc, Nanog, Oct-3/4 and LIN-28). After culturing the iPSCs and if desired adding mutations, the iPSCs can be differentiated in various tissues. https://www.rndsystems.com/resources/articles/differentiation-potential-induced-pluripotent-stem-cells

Now back to one of the applications Martin and his team focus on: treatment of Cystic Fibrosis. Cystic fibrosis is a severe and quite common disease, 1:2000 new-borns is diagnosed with CF. The disease is almost always caused by a single point mutation in the CFTR gene: F508del-CFTR. This mutation leads to a shorter and dysfunctional protein. Martin and his team want to investigate this mutation in iPSCs.

The workflow of Martin and his team goes as follows: first they generate CF specific iPSCs from the peripheral blood of CF patients. For his research, he uses patients with varieties in severity of CF. Next, he wants to pick the interesting clones (in which CFTR is expressed but not functional). Since the known antibodies for CFTR are not reliable, the lab uses a CFTR construct labelled with tdtomato. To screen for functionality, he uses eYFP labelling of the cell. eYFP activity is regulated by the iodide concentration, which is travels in and out the cell through the CFTR channel (figure 2). So when a colony of iPSCs is both fluorescent for the red tdtomato and yellow YFP, Martin can use it for further research. Now, a protocol is needed in which iPSCs can be differentiated in functional lung cells. There is a protocol proposed by Kadzik and Morrisey (2012) for this differentiation. However, Martin wants to use an upscaled version. At the moment, the upscaled procedure is not as efficient as the original variant, but it is still work in progress.

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Figure 2: YFP is sensitive for anions like iodide. It will get activated when iodide enters the cell. Which happens when the CFTR channel is functional.  Adapted from Vijftigschild, L.A.W., Ent, C.K. van der, Beekman, J.M. (2013) A novel fluorescent sensor for measurement of CFTR function by flow cytometry. Cytometry Part A 83A: 578 fig. 1A

Besides CF, Martins research also focusses on iPSCs derived cardiomyocytes as cellular therapeutic for heart repair. In order for therapies like these to exist, cardiomyocytes need to be produced in a safe, efficient and large scale production. In this case large scale production is a real must, since humans lose 1-2 billion cardiomyocytes after a myocardial infarction. Martin and his team are testing these large-scale productions. One important finding is the relationship between the state of differentiation and the density in which the cells where located. This stresses the importance of an equally divide density of cells over the entire tank.

It is now possible to form small amounts of differentiated tissue (few centimeter in diameter), which can beat like heart-tissue. However, the tissue is far from the strength of adult heart tissue. On the bright side, the tissue can deal with higher forces than the heart-tissue of new-borns. At the moment, stem cell derived cardiomyocyte tissue is implanted in monkeys who have suffered a myocardial infarction. This means it is time to prepare for clinical application. The start of ‘iPSCs for Clinically Applicable heart Repair’ or iCARE will help in the realization of clinical application.

I thought this seminar was one of the most interesting ones I have attended. It made me realise how incredible fast the progress in stem cell studies goes. I liked to see the connection between research and clinic. Martin used many clear examples which made his talk easy to follow. I also liked to see the enthusiasm with which he talks about his work. This mainly came back at the end when he got some interesting questions. All in all a good seminar.

 

From Stem Cells to Organs: Exploiting the organ niche for interspecies organogenesis

Speaker: Hiro Nakauchi
Speaker Institute: Stanford University
Organising Department: Developmental Biology
Subject:  From Stem Cells to Organs: Exploiting the organ niche for interspecies organogenesis
Location: Erasmus MC
Date:  24-11-2016

Author: Nemo Andrea          

Hiro Nakauchi is Professor of Genetics Operations at Stanford University. He received his Master’s Degree from Yokohama City University School of Medicine and obtained a PhD in immunology from University of Tokyo Graduate School of Medicine. He did a postdoc at Stanford University. In this seminar, he gave an overview of his research into induced pluripotent stem cells (iPSC) and their applications regarding (interspecies) organogenesis.

His lecture started out focusing on the social relevance of advances in organogenesis technique. It is well known that there is an ever increasing shortage of organ donors. This shortage not only results in many patients being unable to get a transplant they so critically need, but also creates a black market for illegal organ trafficking, resulting in an even greater number of people suffering as a result of organ shortage. He explained how organs grown from the patient’s own cells could resolve these problems. This shows that his research is not just theoretical, but that it is also of great societal relevance.

The discovery of iPSC, as he describes, was a major breakthrough in the field of medicine and biology. It allowed for the generation of stem cells from (theoretically) any somatic cell in the body. While these iPSC can be used in cell therapy to treat specific diseases, they cannot be grown into organs, which are highly complex three dimensional structures. While in theory it should be possible to grow organs fully from iPSC, this is incredibly difficult in practice and may never become a practically attainable option. Hiro and his research group had a solution to this problem that had to be verified experimentally.

The first experiments had as aim to verify the viability of chimeras created by placing iPSC from a donor mouse into a blastocyst of a different mouse. This experiment was successful, as it was found that the resulting mouse was a mosaic of the two cell types. Some cells were from the original mouse and others were of the donor mouse. They decided to take this one step further and one step closer to organogenesis. They made a knockout mouse blastocyst that could not grow a kidney, and once again added the iPSC from a different mouse to the developing blastocyst. They found that the resulting mouse was completely healthy and upon closer inspection they found that the entire kidney was made out of the donor mouse’s cells. Other knockout mice were also successfully rescued from their artificially induced deficiencies.

While these experiments were very promising, one could not conclude whether this would also work for iPSC with xenogeneic barrier. This was also researched by Hiro’s research group and they found that in the same experiment as above, but now carried out with mouse and rat cells still produced viable chimeras and organs. They found that the size of the organism and organs were determined by the species of origin of the blastocyst. They were also able to grow mouse islets in a rat and then transplant those cells into a diabetic mouse and thereby make this mouse recover from its diabetic phenotype.

They were also able to create pig chimeras using the same approach. One big barrier remained: the creating of human iPSC. While human iPSC can be generated, the iPSC that can be created resemble a state later in cell differentiation compared to pig and mouse iPSC. This made chimera formation impossible. At the same time, he mentioned how another problem was also something that had to be resolved: the humanisation of the host animal. It would ethically be hard to grow organs in a half human-pig chimera. He expressed how this could be resolved if it were possible to only have the iPSC create the specific organ desired. Further experiments found a way to allow for chimera formation even out of the (more differentiated) human iPSC. They were even able to allow for chimera formation out of progenitor cells (cells that can only become one cell type/group) which meant they could now overcome both the problem of humanisation, as these progenitor cells can only make a small subset of all body cells (e.g. only liver related cells) and the problem of human chimeras.

This brings us to the current state of research. Research into Human-Sheep chimeras is currently underway. Additionally, one of the reasons why Professor Nakauchi was present to give this talk was because he is here to talk with lawmakers and ethics specialists to discuss how the law should reflect the current state and potential of this technology.

[1] left: diagram of the main concept; [2] right: Graphical respresentation of the zenogeneic organ generation 

I found it very exciting to hear about this research, as it is truly remarkable how well this all works considering the relatively small knowledge we have of exact organ formation and development. I think there is a lot of room for further research in the exact mechanisms governing this transspecies organogenesis. I intend to do part of my Honours Programme on the topic of iPSC, so this topic is of great interest to me. I hope this research will also be able to eventually result in something that can save the many patients waiting for a suitable donor.

[1] http://www.sciencemag.org/news/2015/10/major-grant-limbo-nih-revisits-ethics-animal-human-chimeras
[2] http://www.cell.com/cell/abstract/S0092-8674(10)00843-3

Wnt/ß-catenin signaling controls cell reprogramming (and nucleosomes form clutches)

Bionanoscience Seminar, 08.05.2015

Speaker: Pia Cosma (Center for Genomic Regulation, Barcelona (Spain))

Author: Edgar Schönfeld

Pia Cosma’s research is focused on cell reprogramming. Her talk was divided into two major subjects that deal with cellular reprogramming: Wnt/ß-Catenin signaling and chromatin organization. Reprogramming can be achieved via nuclear transfer or direct reprogramming, which involves the introduction of the 4 pluripotency factors Oct4, Sox2, cMyc, and Klf4 into a somatic cell. A third option is reprogramming via cell fusion. Cell fusion itself is a natural process, which occurs for instance during the differentiation of muscle cells. However, cells can also be fused artificially in the lab, with the help of chemicals or electricity. If you fuse a stem cell with a somatic cell they give rise to pluripotent hybrid cell, because the pluripotent genome is dominant over the somatic one. (This process also happens in vivo and is involved in wound healing.) After such a fusion the pluripotency factor Oct4 is reactivated. In an experiment Pia tagged Oct4 with GFP to observe spontaneous cell fusions. She found out that reprogramming only occurs when the right amount of ß-Catenin is present, which is activated by the Wnt pathway.

In the last years she discovered a lot more details. She discovered that the ß-Catenin concentration fluctuates in embryonic stem cells (ESCs), which is absolutely important for the reprogramming of somatic cells via fusion with ESCs. Different levels of activity of ß-Catenin can regulate either pluripotency or differentiation. Furthermore she found out that repression of TSF3, a regulator of the Wnt pathway, can increase the reprogramming frequency several hundred times.

With that in mind, she wanted to know what happens to chromatin organization during reprogramming. As a result the textbook model of chromatin packing needs an important update! To be precise, the 3rd panel from the left in figure 1 is inaccurate. Pia discovered (using STORM) that the nucleosomes are not distributed uniformly over the genome. Instead nucleosome aggregate in small or big groups, which Pia calls ‘clutches’ (Fig.2).

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Figure 1: DNA Packaging, taken from http://www.shmoop.com/dna/dna-packaging.html

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Figure 2: New Model, source: http://www.cell.com/abstract/S0092-8674%2815%2900132-4

These clutches are separated by stretches of nucleosome-free DNA. Thereby the larger clutches form the silenced heterochromatin, because they contain more of the linker histone H1. RNA polymerases can still associate with the small clutches. In stem cells and during reprogramming the DNA is mostly covered with small clutches, which facilitates the docking of transcription factors and RNA polymerase. In somatic cells however, DNA is mostly packed around big silencing clutches. In an reprogramming event the cell has to switch from big dense to small loose clutches, but how is this achieved? Pia’s team hypothesized that this might occur through nucleosome sliding and removal, which is in accordance with their simulations. To summarize their findings the team created this video: