Print Page   |   Contact Us   |   Sign In   |   Become a Member
Site Search
Society News
Blog Home All Blogs
Search all posts for:   


View all (24) posts »

Webinar Review: “For singles only”, the new admission criteria in the hematopoietic stem cell biology field.

Posted By Connections Editor, Wednesday, December 23, 2015

Eugenia (Kena) Flores-Figueroa. Oncological Research Unit at the Mexican Institute of Social Health, Mexico City, Mexico.


In the last few years we have witnessed the shift from studies addressing heterogeneous hematopoietic stem cell (HSC) populations to single cell studies, which have contributed to a better understanding of the clonal heterogeneity of stem cells, the dynamics of stem/progenitor cells over time and upon inflammation, their location within the bone marrow (niche) and their hierarchy.  Technical innovations to label and track single cells, is the force reshaping stem cell biology. These new technologies might look intimidating and hard to follow for the non-expert (like me), but it is definitely a must-do for anyone on this field. To my delight, ISEH recently organized their first technology-focused webinar on this topic, “comparison of methods for cell labeling and clone tracking”, which helped me to get a better understanding of the methodologies, and also of the field as a whole. Interestingly, I had a completely different - but equally useful - experience compared to the recorded webinar; the live webinar was more interactive and I had the chance to ask a question to one of the speakers, and the recorded webinar gave me the chance to set my own pace, read the papers cited by the speakers, and reach a much deeper understanding.

Dr. David Kent, from the University of Cambridge, was the first speaker. He explained the problem faced by the stem cell biology field using an analogy from his former mentor; “If you have two handfuls of sand, one in each hand, and only one has a seed in it, in order to know which handful of sand has the seed, you will have to plant both; so you will know that the handful of sand capable of growing a plant had the seed. The problem is that if you are interested in studying the seed, after the assay the seed no longer exists, as it has become a plant, and if you study the handful of sand before you plant it, you will end up describing mostly sand and not seed”. So how can you study the molecules in a seed? In the analogy, the seed represents a HSC, and the test that helps us find out if the handful of sand is a plant, is the gold standard in the stem cell field, the transplantation of single cells into an irradiated host, followed by engraftment analysis using donor cell specific cell surface markers (like CD45). The problem with this approach is that the assay to test whether or not a cell is truly stem cell had to be measured by the progeny of the cell, thereby destroying the original cell. In this technology-focus webinar, Dr. David Kent and Dr. Leila Perié, from the Curie Institute, discuss two different approaches to overcome that problem, chaired by Dr. Claudia Waskow, from the TU Dresden.

Dr. David Kent adapted a flow cytometry technology called “Index Sorting” to integrate single cell gene expression and single cell functional data. His approach was to transcriptionally profile single cells from several cell isolation strategies (from many different groups) to identify the genes that overlapped between them with the rationale that the non-overlapping cells would represent different contaminating cells, and the overlapping genes will identify stem cells. Then he performed functional assays, and assigned individual outcomes, and coupled these data by assigning an individual outcome to each of the cells and retrospectively plotting those outcomes on a flow cytometry plot that also contained the gene expression outcomes (Wilson et al., 2015, Cell Stem Cell & Schulte et al., 2015, Exp Hematol). Using this strategy, Dr. Kent’s laboratory was able to identify a population that contains 50% of murine HSC function using the index sort data and track the markers CD150+ CD48- SCA-1 high EPCR+ that were less committed, divided slower, retained HSC markers, showed higher chimerism and robust multilineage progeny. His pioneering method has made it possible to study single cell biology and link mammalian stem cell functions with different markers and pathways. After his presentation, Dr. David Kent was approached by many interesting questions from the audience, where he discussed the limitations of his technique and recognized that there are still unanswered questions like the symmetric vs asymmetric division of HSC, where data is not conclusive; the dependence of transplantation models that still have technical challenges, like the time of engraftment read out, that have increased from  12, 16 , 24, 32 weeks, but that it may still not include rare stem cells that repopulate mouse after 6 months. Index Sorting is a technique that can be applied to any population where there is a defined single cell assay, this technique has been successful in the study of human mammary progenitor cells, but studying human HSC are still a challenge.

Dr. Leila Perié’s barcoding technique to track single cell lineages has allowed her to decipher, reshape and “trim” the hematopoietic tree to redefine the hierarchy of different progenitors (Naik S., 2013, Nature; Perié L., 2014, Cell Rep; Naik SH., 2014, Exp Hematol; Perié L., 2015 Semin Cell Dev Biol). In the webinar, Dr. Perie presented a global view of lineage tracing methods and offered a guided step-by-step list to choose the right method for each project and then focused on the barcoding technique and gave some useful examples of her discoveries using this technique. After her presentation, I had a clearer vision of where to start and which questions I should address first. The barcoding technique relies on the “insertion” of a unique sequence using a viral library (the barcode) into your cell of interest, the transplantation of those cells (on a murine system) and then tracking the barcode (usually by nested PCR and deep sequencing). Dr. Perié’s advice was to first ask ourselves if our population of interest can be transduced, this is a critical step and the biggest limitation of this techniques (for example, human and leukemic HSC are difficult to transduce); if we choose a retrovirus, we will need at least one cell division to integrate the barcode, which is not the case for a lentivirus, were we optimized the time for transduction (usually 6 hours). The diversity of the library, the size of the barcode and the sequence is another critical step, and finally the bioinformatics step, where there is no standard method.

The barcoding technique is not free of challenges like the repeated usage of the same barcode within two different cells, multiple integrations, low transduction efficiency and size of the cell expansion, among others. Dr. Leila Perié’s own work demonstrates the power of this technique, when you face those challenges, as she has contributed to the literature of tracing the fate map of LMPP, MDB and CMPs analyzing their heterogeneous outcomes , which has helped to redefined the hematopoietic tree, regarding dendritic, erythroid and myeloid cells.

The analysis of single cell biology has dramatically reshaped our field, not only addressing questions that we were not able to before, but also changing old paradigms, new and young laboratories are contributing to power this revolution, and I encourage each member of our society and a member of our field to keep up with these novel techniques; webinars are one of the tools we have to learn and to discuss these methods. There are still many unanswered questions, and I think this webinar will encourage many of our young scientists into this field. 

This post has not been tagged.

Share |
Permalink | Comments (0)
Association Management Software Powered by®  ::  Legal