Blog Comment contest 10-31 May ’12: HSC terminology

Hereby, we announce the beginning of the Maximow Award Phase II – to participate, simply leave a comment!

Question:
What exactly do we mean when we talk about “hematopoietic stem cells”, how sure are we about this and is this terminology for HSC adequately dealt with in different languages?

More information, including eligibility and prize money, and a translation of the question into Russian and German can be found in the post of April 20th.

Good luck!

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15 Responses to Blog Comment contest 10-31 May ’12: HSC terminology

  1. dontworry says:

    Right now, this topic of the Maximow contest is quite hot. Too many people working in biomedicine use the term “stem cell” in a very wide sense. In many cases, this term is extended to many cell populations that may be ineffective and even dangerous to the patient receiving cell therapy.
    Meanwhile, the word “stem cell” simply means an ancestor blood-generating cell occuring in early embryonal life of higher animals. The term “stem cell(s)” was born in hard discussions about common predecessors for different blood populations. A number of famous (mostly, German) scientists, from R. Virchow to A. Pappenheim and A. Maximow, had in mind the concept of a stem cell (last 1/3 of XIX century). The only issue was to identify and characterize these predecessors of mature blood cells.
    This proved to be possible only by new methods of differential cell staining and advancement in developmental biology, during the first decade of the 20th century. Later, throughout the next 100 years, the term “stem cell” in blood research did not change its meaning as a seminal element of a phylogenetic tree. By now, however, it is very much better defined since specific biological markers are known for every step of stem cell differentiation, from Maximow’s “large lymphocytes” resident in hemopoietic organs to a variety of quite mature red and white cell populations scattered in our tissues. Hence, I hypothesize that the discussion may address comparative properties and biomarkers of stem cells at initial/very early stages of their growth and differentiation.

    doi:10.3205/maximowaward_2012_blog-comment_01

    Please cite this contribution as follows: dontworry. Right now, this topic of the Maximow contest is quite hot. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_01. doi:10.3205/maximowaward_2012_blog-comment_01

  2. Fascinating news on stem cells, still a lot to explore in this field.
    However, I can’t see any terminological pitfalls here. We can leave the term ‘stem cell’ as it is, to name a general subject, a ‘stem’ term as such, and then think about new, more precise terms for recently discovered subpopulations.
    I think some authors have already done this for us – see Muller-Sieburg et al., March 2012, Blood. They say: “Three subsets can be distinguished when HSCs are classified by their differentiation capacity: myeloid-biased (My-bi), balanced (Bala), and lymphoid-biased (Ly-bi) HSCs”. Why not to use those terms?
    Correct me if I’m wrong.
    Thanks!

    doi:10.3205/maximowaward_2012_blog-comment_02

    Please cite this contribution as follows: Yana Onikiychuk. Fascinating news on stem cells, still a lot to explore. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_02. doi:10.3205/maximowaward_2012_blog-comment_02

  3. Ilya says:

    Dear Colleagues!

    Despite the seeming triviality of the issue and excessive theorizing, it is very important. Often, medical (biomedical) concepts, principles and terms are very different even in various cities within a country. I am sure that terminological differences between the scientific communities of different countries is even more significant.
    Before talking about the HSC, I’ll start with the other example, which is even more striking, in my opinion. There are two types of cells (“pericytes” and “perivascular cells”) in the nomenclature of the Russian histology school. Perivascular cells are located around the vessels, these cells are also called “adventitial cells”. In contrast, pericytes are located into the wall of blood vessels closer to the intima, where contact with endothelial cells. It is completely different types of cells in understanding of Russian scientists. However, some researchers from other countries often equate these two terms.
    To answer the question about interpretation of the term “hematopoietic stem cells” (HSC), we must first define the concepts and principles of histological schools in different countries. So, the concept of “cellular-differon organization” (клеточно-дифферонной организации) is well recognized in Russia (you can believe me because I am from Russia). I can explain this concept in a few words: “a life is continual”. I’ll try to expound. Life is constantly moving, regardless of our mood, worldview, perceptions, irrespective of the moment in which we look at it, evaluate and interpret it. If today you saw a person in a suit, it does not mean that he will wear it again tomorrow. He can wear a coat or shirt, depending on the weather or other reasons. He can changes an appearance or even a sex . However, there are absolutely immutable things. Namely, a biological sense any man has initial (a zygote) and end points of development.
    As for the cells, the situation is similar. “Cell differon” is a continual cytogenetic line of single cellular type from “stem” to a “specialized” cell. In other words, each cell goes a way of development, during which the constantly changing its properties and functional characteristics. Life of cells are continuous and proceeds from the initial point (“stem cell”) to the end point as well. We can identify its characteristics only in concrete moment of time when we perform some studies (as though we have stopped time). As all types of stem cells (the initial points) can be counted on the fingers and there are a lot of specialized cells types it is quite clear that one type of stem cells can gives rise to different cellular differons.
    In this regard, HSC is a population of stem cells which gives rise to differons of the all blood cells (and other differentiation pathways was described but the main is hematopoietic). Thus, HSC is the initial point. There are a lot of opportunities to study the HSC population and to divide into many subpopulations by the use of dozens methods, hundreds of immunophenotypic antibodies, etc. But in my opinion, it is a conceptual error. It is important that the HSC as the initial point (true stem cells) of several cellular differons can not have a determination for differentiation into any cell lines. True HSC should be “neutral”. In this regard, some terminological hypothesis are debatable. For example, Yana Onikiychuk (post above) proposed to use the terminology of Muller-Sieburg et al. (2012), which consists in dividing HSC into three subpopulations by their differentiation capacity: myeloid-biased, balanced, and lymphoid-biased HSCs. However, from the perspective of the classical school of histology two of these subpopulations are not HSCs because these cells already have a bias toward one of differons. What does it means? It means that these cells have already left the the initial point and move to the end point. Its can be called such as “undifferentiated precursors of myeloid or lymphoid cell lines”, but not HSCs. At the same time, the population was called “balanced” by Muller-Sieburg et al. more consistent with the true HSC.
    Thus, the term “HSC” has a conceptual significance. It means a single self-renewing population of cells which possess capacity to give rise to different types of blood cells (first of all) equally. Close derivatives of HSC (cells that have been starting differentiation already) could be attributed to HSC population due to poor technological opportunities at the time of Maximow, for ex. Possessing modern technologies it is important to remember the fundamental concepts and distinguish between the true HSCs and their immediate descendants which nowise should be considered as a subpopulations of HSCs.
    Sorry for my bad English .

    doi:10.3205/maximowaward_2012_blog-comment_03

    Please cite this contribution as follows: Ilya [Bozo]. Dear Colleagues! Despite the seeming triviality of the issue and. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_03. doi:10.3205/maximowaward_2012_blog-comment_03

    • Kshama Gupta says:

      Dear Colleagues,

      I believe that this is a topic on the usage of the term HSC, being biased medically, scientifically and of-course, hence forth regionally, is a topic of immense debate and discussion.

      Foremost, we all would agree and appreciate that a hematopoietic STEM cell conceptually, is the one having ability to contribute to all the cell lineages of blood.

      The intensive search for the population of such cells having the ability to self renew and repopulate began right after the disaster of Hiroshima and Nagasaki in 1945. In 1956, three laboratories demonstrated that the injected bone marrow cells directly regenerated the blood-forming system, rather than releasing factors that caused the recipients’ cells to repair irradiation damage. To date, the transplantation of these Hematopoietic stem cells, is the only known treatment for Bone Marrow Failure syndromes and to regenerate the blood forming system.

      However, this population of cells we term the “HSC” medically does not comprise the pure population with the homogenous property of “Stemness” (i.e. Self – Renew and re-populate the various Hematopoietic lineages), and is rather a mixed population of Hematopoietic stem / progenitor cells at different stages of differentiation.

      A major thrust of basic HSC research since the 1960s has been in identifying and characterizing the true stem cells. The challenge is formidable as only about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell. In the blood stream the proportion falls to 1 in 100,000 blood cells.

      Further more its known that the short-term blood-progenitor cells in a mouse may restore hematopoiesis for three to four months (Harrison et al) and the longevity of short-term stem cells for humans is not firmly established. A true stem cell, capable of self-renewal, must be able to renew itself for the entire lifespan of an organism. It is these long-term replicating HSCs that are most important for developing HSC-based cell therapies. Unfortunately, to date, researchers cannot precisely distinguish the long-term from the short-term cells when they are removed from the bloodstream or bone marrow.

      In 1988, in an effort to develop a reliable means of identifying these cells, Irving Weissman and his collaborators focused attention on a set of protein markers on the surface of mouse blood cells that were associated with increased likelihood that the cell was a long-term HSC. Four years later, the laboratory proposed a comparable set of markers for the human stem cell. However, the groups of cells thus sorted by surface markers are yet heterogeneous and include some cells that are true, long-term self-renewing stem cells, some shorter-term progenitors, and some non-stem cells. Although it has been shown that a purified population with surface markers as CD34+, Thy1+ and Lin- swiftly engraft, yet a great deal of re-consideration is required when we talk about Stem Cells in Hematopoietic system. Moreover, to isolate mesenchymal stem cell with respect to the hematopoietic cell population from bone marrow has been another challenge due to certain overlapping markers.

      In agreement with the post by Ilya, I would also say that the use of the terminology by Muller-Sieburg et al. (2012), which divides the HSC into three subpopulations by their differentiation capacity: myeloid-biased, balanced, and lymphoid-biased HSCs, however does not follow the classical definition of Stem cells for two of the sub-populations included; Being already committed to follow one of the lineages down stream, these would no longer give rise to all the Hematopoietic cell types. There are several research groups working on the differentiation models (ie. Stochastic, Determinant, Selective or Instructive – Lineage specific, or Combination Models) so as to address the issue of heterogenecity in the population of HSC and their differentiation paths taken under the influence of the niche / microenvironments / stimuli; A mixed population of CD34+ cells in presence of a particular cytokine / Growth factor would all majorly behave in a similar manner, while in presence of a combination of different stimuli would chose to diverge and differentiate to multi-lineages. Hence to classify the pre-committed cell types as progenitors with a mixed population as stem – progenitors, giving appreciable consideration to the sub-populations derived from the actual HSC, would be more appropriate. We hope with the ongoing advancements in identifying and isolating the pure population of LT-HSC or its immediate descendants at a single cell level would open new avenues for a further precise distinction and classification of the Hematopoietic stem / progenitor repertoire.

      doi:10.3205/maximowaward_2012_blog-comment_10

      Please cite this contribution as follows: Kshama Gupta. Dear Colleagues, I believe that this is a topic on. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_10.
      doi:10.3205/maximowaward_2012_blog-comment_10

      • Alexey says:

        Kshama,
        We don’t consider the “biased HSC” as pre-commited cells, because they retain multilineage repopulation capability. Now it’s clear for myeloid-biased HSC. Both Nakauchi (Morita 2010) and Eaves groups (2007, 2009, 2012) confirmed that myeloid-biased HSC can give robust multilineage engraftment in secondary transplant. It still not clear for lympoid-biased (Morita 2010 – yes, Eaves group – no or under?), we need more data. So, it’s not black and white.

        In terms of medical practicality to call it “HSC”. Even though, in the clinic, we always use a mix stem and progenitor cells, we can still confidently use and call it HSC transplant/ therapy if therapeutic effects underlie HSC function and if we can detect donor-derived multilineage repopulation and engraftment for many years or life-long. In many cases in hematology-oncology we don’t need donor’s HSC function, we need progenitor cells for resque ablated marrow, save a patient from neutropenia and we need T-cells for graft-versus-tumor effect. So, sometimes hematology-oncology patients can get rid of disease without donor’s HSC. In this case you can call it “cell therapy” or “progenitor cell therapy” or “T-cell therapy”, depends on mechanisms. Frequently stem cell labs in the hospitals label a graft as “HPC” product = “hematopoietic progenitor cells”.

        In terms of HSC markers, CD34 is only on of HSC markers for human, but mouse CD34+ will not engraft long-term. And with SLAM situation is quite opposite – in mouse LSK/SLAM (or E-SLAM) are HSC, but in human SLAM HSC are progenitors. You can read here –
        http://stemcellassays.com/2011/04/the-brief-history-of-phenotype-dissection-of-hematopoietic-stem-cells/
        and
        http://stemcellassays.com/2011/05/the-brief-history-of-phenotype-dissection-of-hematopoietic-stem-cells-an-update/

        doi:10.3205/maximowaward_2012_blog-comment_12

        Please cite this contribution as follows: Alexey [Bersenev]. Kshama, We don’t consider the “biased HSC” as pre-commited. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_12. doi:10.3205/maximowaward_2012_blog-comment_12

      • Ilya says:

        Alexey,
        I see you are quite well versed in different methods of HSC processing in vitro. Despite this, you take the discussion away from the main issue which was indicated in the title of blog. There are not questions regarding the methodology of cell processing, more precisely, these questions are minor. Characteristics of HSCs populations is the main point of our disagreement.
        In my opinion a true HSC is the initial point for all differons of blood cells (and some other kind of cell, for ex. fibroblasts, etc.). I do not exclude the possibility that HSC population is heterogeneous. However, I am against the separation that you indicated and called as generally accepted.
        You said: “We don’t consider the “biased HSC” as pre-commited cells, because they retain multilineage repopulation capability”. Then please tell us what are you talking about “bias” if each of these subpopulations have multilineage repopulation capability? So, it have self-renewal, multilineage differentiation, and a similar pattern of markers. I said earlier about this contradiction in your words.

        doi:10.3205/maximowaward_2012_blog-comment_14

        Please cite this contribution as follows: Ilya [Bozo]. Alexey, I see you are quite well versed in different. Blog comment, Maximow Award contest, June 2012. Cell Ther Transplant/Maximow Award, June 2012;blog-comment_14. doi:10.3205/maximowaward_2012_blog-comment_14

      • Alexey says:

        Ilya,
        I didn’t name them “biased”, but research groups who discovered them did. And they did so, because they saw a very different pattern of population after primary transplant of single HSC. So, they decided to describe this biological phenomenon in this way (“biased HSC”). When I said that there is an agreement that biased HSC are stem cells but not commited progenotors, I based my opinion on (1) conclusions made in well known HSC heterogeneity studies, (2) commentaries to these studies published by experts, (3) talks from conferences and (4) personal communication (for instance with people from Eaves group). If you’re saying that there is a contradiction and diagreement in the field, plese link to it. I don’t remember I saw any contradictory and negative comments about these studies conclusions. If you’re expressing your personal opinion, I’m fine with it. I told your mine, which is in agreement of Eaves and Nakauchi groups.

        I don’t think I discussed offtopic things, because all conclusions about HSC definition and heterogeneity based on aexperimental assays and their interpretation. If we don’t understand them, we will discuss about different thing on different languages.

        PS: I’m sorry but forum platform doesn’t allow me reply under your comment precisely

        doi:10.3205/maximowaward_2012_blog-comment_13

        Please cite this contribution as follows: Alexey [Bersenev]. Ilya, I didn’t name them “biased”, but research groups who. Blog comment, Maximow Award contest, June 2012. Cell Ther Transplant/Maximow Award, June 2012;blog-comment_13. doi:10.3205/maximowaward_2012_blog-comment_13

  4. Alexey says:

    The current definition of “stem cell” include 2 essential characteristics: the cell which (1) capable to self-renew (or reproduce itself) and (2) capable to give many (pluri- or multi- potency) mature cell types (lineages). Although this definition causes some discussion, especially among embryonic stem researchers, that’s the best we have today. Yet another hallmarks of adult stem cells include: niche, specific cell cycle and regenerative potential (ability to heal or rescue missing cell types).

    Hematopoietic stem cell (HSC) fits perfectly in this definition. We can assess self-renewal capability of HSC and easily measure their multipotentiality. Bone marrow transplantation and enriched purified HSC transplantation allow us to assess multipotentiality in vivo and quantify the number of functional HSC. Serial transplantation (from mouse to mouse) allow us to track single HSC and therefore provide an evidence for self-renewal. This is functional definition. There is a weak correlation with particular HSC markers due to high heterogeneity of their population. For example, surface markers allow to enrich some HSC subpopulations, but not guaranteed functional performance. Thus, only one out four highly purified (based on 6-8 markers) human HSC will self-renew and possess multipotentiality in xenotransplantation model. Because we can not assess self-renewal in human, we can define HSC after transplantation by life-long multilineage engraftment in bone marrow and chimerism in the blood.

    The problem of HSC heterogeneity was studied in details in the last few years. The current concept implies co-existance of few sub-populations (or clones) HSC with different patterns of lineages repopulation (-potentiality): myeloid-, lymphoid- biased and balanced. Existence of these clones doesn’t cancel HSC definition, because all of them able to self-renew, means engraft and repopulate in serial transplantation assay. These HSC subtypes could also have different cell cycle status and kinetics. So, adult HSC is a dynamic population, which consist of different clones with different blood repopulation patterns and equilibrium of quiescent and cycling states. Now we’re getting close to realization that adult HSC can’t not give absolutely all (100%) of different blood cell types. Some of adult HSC can never give tissue macrophages or some B-cell subsets. According a current hypothesis, these blood types have developed as embryonic remnants from embryonic HSC.

    So, in the definition debate, HSC could serve as good example of how “stem cell” can be defined and measured in assays.

    doi:10.3205/maximowaward_2012_blog-comment_04

    Please cite this contribution as follows: Alexey [Bersenev]. The current definition of “stem cell” include 2 essential characteristics: Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_04.
    doi:10.3205/maximowaward_2012_blog-comment_04

    • Ilya says:

      I still do not agree with Alexey. If HSC has focus in a particular direction of differentiation (myeloid-, lymphoid- biased, etc.), then it should not be considered HSCs, but as closest of its derivatives. Of course, the minimal but sufficient for clinical use methods of obtaining HSC does not allow us to accurately separate the true HSC from their closest derivatives. Moreover, for routine clinical practice such separation may not be necessary. However, in general biological sense, the population of HSCs are still more homogeneous.
      All terminological problems are caused by different interpretation of new data. New data (in respect of immunophenotype, differentiation potential, etc.) can be used for division of HSC to a lot of subpopulations or for purification of HSC from the nearest derivatives. As for me, I like the second option, which is more correlated with the terms of the classical school of histology.

      doi:10.3205/maximowaward_2012_blog-comment_05

      Please cite this contribution as follows: Ilya [Bozo]. I still do not agree with Alexey. If HSC has. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_05. doi:10.3205/maximowaward_2012_blog-comment_05

      • Alexey says:

        Ilya,
        My summary based on experimental data, published intensively in the last 7 years. These data indicate to high degree of heterogeneity in adult bone marrow HSC population. I’ll give you few examples and references.
        If you take a single HSC and transplant it, you can see different patterns of repopulaion in primary recipients: myeloid-, lymphoid- biased and balanced. The ultimate test for “stemness” is serial transpant. So, if we look at secondary transplant, we can see that balanced HSC can give balanced or myeloid- biased repopulation, myeloid-biased HSC can give balanced or myeloid-biased repopulation and lymphoid biased HSC can give balanced or lymphoid-biased repopulation in secondary recipients. So, balanced HSC can become myeloid-biased, but the most typical case is the opposite – when in primary recipient we can see only myeloid repopulation, but in secondary – robust multilineage. All of these HSC subsets passed self-renewal test and therefore we can call them “stem cells”, based on current international consensus. If myeloid-biased HSC is not really “stem” but “commited progenitor” (as you’re saying), they will not self-renew or will give only myeloid repopulation in secondary recipient. It’s not the case in single cell transplantation assays. You have to understand that we can clearly separate HSC from their progenies by transplantation assays, but not by markers. Please read: Dykstra 2007, Morita 2010, Benz 2012. This is a current consensus between many groups, studying HSC around the world – Connie Eaves, Nakauchi, Goodell and others. They performed as many as few thousand of single cell transplants and published about 10 papers on this subject. One can argue that “biased HSC” are commited progenitors, but experimental data indicate to opposite – they retain multilineage repopulation capacity throughout lifespan.

        I’m not sure to what authors and time you’re referring when talking about “classic histology”. Stem cell field is so dynamic right now, that some chapters of textbooks should be revised every 5-10 years or so. For example, still 3 years ago we had a dogma in the field that all HSC are quiescent and not cycling. But now we have got some evidence, that HSC can go to cycle and quiescence back and forth, depending on necessity and feedback signals. One quiescent HSC could undergo 5 divisions and then become quiescent again, retaining the ability to self-renew and reconstitute many blood lineages.

        So, all HSC subsets reside in dynamic equilibrium. HSC heterogeneity makes a lot of biological sense.

        doi:10.3205/maximowaward_2012_blog-comment_06

        Please cite this contribution as follows: Alexey [Bersenev]. Ilya, My summary based on experimental data, published intensively in. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_06. doi:10.3205/maximowaward_2012_blog-comment_06

      • Ilya says:

        Alexey, do you think, before I formed my point of view, I have not read the experimental and clinical data of the last ten years? I certainly know the researchs of respected scientists, who were listed in your comments. However, interpretation of the results of these studies is not so clear. I would have explained more precisely in Russian, but I’ll try in English :).

        You said that ‘the ultimate test for “stemness” is serial transpant”. However, it is not. The ultimate test for “stemness” is clonal analysis, in which the researcher obtains from a single cell whole colony of cells, it is absolutely identical to the primary cell (this is a classic proof of self-renewal). While the method of serial transplantations is characterized by manipulating with groups of cells. It is indicated in the articles which you have listed.

        Now define the meaning of the term “heterogeneity”. First of all, this concept represents a different origin. However, in the scientific literature, the term “heterogeneity” has a wider meaning and is used when we select any subpopulations of cells (in this case, the cells may have the same origin, but significantly (and consistently) different from each other by phenotypic and functional features). Do you agree?

        Now back to your examples. You have written:
        “If we look at secondary transplant, we can see that balanced HSC can give balanced or myeloid- biased repopulation, myeloid-biased HSC can give balanced or myeloid-biased repopulation and lymphoid biased HSC can give balanced or lymphoid-biased repopulation in secondary recipients.”

        I consider that enumeration of these results deny your own opinion. I will try to illustrate the idea by the following examples. We have two criteria, namely a capacity of differentiation and self-renewal (two variables). So, if in serial transplants lymphoid-biased HSC (for example) give lymphoid-biased repopulations always and have a self-renewal, they would have to be considered as a specific “subpopulation” of HSC. Another option: lymphoid-biased HSC give a lymphoid-biased repopulations always in serial transplantation, but have not a self-renewal. In this case, these cells should be called “the closest derivatives” of HSC (it is not a “stem cells” already). The third option corresponds to your examples: lymphoid-biased HSC can give lymphoid repopulation in primary recipients, but it can give balanced or lymphoid-biased repopulation in secondary recipients and have a self-renewal. What does it mean? In my opinion, this means only one thing … This subpopulation (lymphoid-biased), as well as myeloid-biased and balanced are not “subpopulations”, it is a single population of HSCs. The changes of differentiation potential, which formed the basis for separation of subpopulations, are transient and highly conditional. The specificity of microenvironment causes transient changes in differentiation capacity of HSCs population. The factors of microenvironment may be different in composition and intensity in different areas of bone marrow stroma even within a single bone (eg, ilium). It is therefore logical that the cells within a population of HSCs differ from each other. But these changes are not stable, it is caused by factors of microenvironment, which act at the specific period of time. In the end, there are not two identical cells within a single population. However, it does not mean that it is necessary to separate populations in a number of 1000 cells (for example) to 1000 subpopulations. In this interpretation, it becomes apparent why we observe a prevailing any differentiation (lymphoid or myeloid) in case of primary recipient, and another differentiation in the secondary recipient (it is another organism with the other factors of microenvironment).

        P.S. Of course, textbooks should be revised every 5-10 years (even more often) but the classic works of Maximow, Zavarzin, Khlopin, etc. are called “classical” not without purpose… It is relevant to this day.

        doi:10.3205/maximowaward_2012_blog-comment_07

        Please cite this contribution as follows: Ilya [Bozo]. Alexey, do you think, before I formed my point of. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_07. doi:10.3205/maximowaward_2012_blog-comment_07

  5. Alexey says:

    Ilya,
    I have nothing against your interpretation of these studies. I agree, everyone can interprets it in different ways. I’m personally with agreement of all conclusions from the studies on HSC heterogeneity done by Eaves and Nakauchi groups.

    What do you mean under “clonal analysis”? What assay? Lineage tracing? Colony assay? What classical assay for HSC self-renewal are you talking about? Can your give a reference? Lineage tracing give as mostly information about stem cell fate, division and differentiation history. In transplantation assay your can measure all of these parameters. Serial transplant is not manipulation of group of cells. As I told you, in primary, you transplant a single cell. You can track exactly the same HSC (equal to cell-ancestor) in bone marrow, sort them and transplant again. But we don’t need to do that for following transplants, that’s why we take whole marrow started from 2nd round.
    Please give an example, how “clonal analysis” would assess HSC self-renewal better than transplantation assays.

    I don’t see where I denied my own opinion. I said if cell passed serial transplant we call it “stem cell”. I understand if someone disagree with this definition. Every assay have limitations.

    In terms of heterogeneity definition. There is no such definition set in HSC field. But everybody understand what other people are talking about. In our case heterogeneity refers to functional differences between phenotypically similar types of HSC. For example, Lin-/cKit+/Sca+/CD34-/CD48-/CD150+ HSC could have few different patterns of repopulation, different cell cycle status and kinetics, different gene expression profile. You can figure out the whole heterogeneity things when you start to do single cell assays. Because the whole population (mixed) HSC could behave in the same manner.

    You can call HSC as one population, which consist of few functionally different subsets (subpopulations/ clones) or call it as different HSC populations. It doesn’t matter. I’d disagree that HSC transplant assay is highly conditional. if it would be, we will see heterogeneity even in primary transplant of the whole marrow, without HSC purification. We use a special mice – genetically identical, same age, hosted in pathogen-free conditions, undergo exactly the same conditioning and receiving the same post-transplant care – to ensure minimal influence of extrinsic factors. Also, I think, people have done some work which indicate that HSC heterogeneity is intrinsic and not depend completely on extrinsic factors (need to double check this).

    doi:10.3205/maximowaward_2012_blog-comment_08

    Please cite this contribution as follows: Alexey [Bersenev]. Ilya, I have nothing against your interpretation of these studies. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_08.
    doi:10.3205/maximowaward_2012_blog-comment_08

    • Ilya says:

      Alexey,
      I’ll answer in order:
      1. You said: “What do you mean under “clonal analysis”? What assay? Lineage tracing? Colony assay? What classical assay for HSC self-renewal are you talking about? Can your give a reference?”
      I have meant a “colony assay”. I’m not going to go far for the references. For example, it is the Morita’s acticle (2010) mentioned by you earlier.
      2. You said: “As I told you, in primary, you transplant a single cell”.
      First, theoretically, the method implies the transplantation from one to ten or more cells. Secondly, in practice it is more than one cell always. Explain to me how can get the expansion of HSCs after transplantation of a single cell? The most HSCs does not reach into stroma and “die” even when it is a therapeutic transplantation in clinical practice (these are the words of professor Afanasiev, V Annual International Symposium “Actual issues of gene and cellular technologies”).
      3. You said: “Please give an example, how “clonal analysis” would assess HSC self-renewal better than transplantation assays”.
      Clonal analysis allows us to manipulate a single cell, but serial transplantation does not allow to do it.
      4. I do not exclude that the population of HSCs ever can be divided into subpopulations. However, the criteria for separating on subpopulations must be very substantial and have a practical sense. Stable functional differences or identified differences of origin can be considered as such criteria. If we see that in some conditions cells have some characteristics and these same cells have other characteristics in other conditions, it is absolutely normal. This is not a reason to divided its into subpopulations.
      You said: “We use a special mice – genetically identical…” Is it the clones? if it is not, then there is no genetic identity too.
      Thanks for an interesting discussion.

      doi:10.3205/maximowaward_2012_blog-comment_09

      Please cite this contribution as follows: Ilya [Bozo]. Alexey, I’ll answer in order: 1. You said: “What. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_09.
      doi:10.3205/maximowaward_2012_blog-comment_09

      • Alexey says:

        Ilya,
        Colony assay is not assessing stem cells, it’s assessing progenitors, unless you do single cell. But, even if you put a single cell sorted as “HSC” you don’t know is it a real stem cell or not without transplantation. Markers are not always translate to function. Remember, in the best case, 1 out 4 sorted cells will repopulate a mouse. So, we never use colony assay to make conclusion about HSC. Culture (in vitro) is very much artificial system and alter HSC function. That’s why golden standard to assess HSC (self-renewal and multilineage differentiation) is in vivo (transplant) assays.

        How would you assess self-renewal in colony assay? The most sophisticated method could include seeding of single cell, get a colony 1 week after, replate this colony (or single stem cell from this colony) to other well and get secondary colony 1 week later. Do a few cycles of re-plating until HSC exhaust. But currently nobody is doing that, because transplantation assays are more trusted and commonly accepted. So if you will try to “sell your story” about HSC function based solely on colony assay, you will never able to publish it. Any reviewer will ask for transplantation assay. Colony assay could be used only as an additional (to transplantation) method to confirm progenitors performance. Transplantation assays can cover absolutely all possibilities on colony assay. You can check any lineage maker, derived from donor’s HSC, you can measure self-renewal nd quantify HSC frequency.

        The reason why Morita did it, was not HSC self-renewal assessment, but rather additional method to confirm/ reject in vivo data. In fact, they didn’t check Meg/erythro- repopulation in translant assay and they did so in colony assay.

        To your second point. You don’t need to do single cell transplantation to assess HSC function. So, we frequently don’t do single cell. I personaly done 3, 10, 30, 100 sorted cells or 3k, 10k, 20k, 50k, 100k, 200k, 0.5M, 1M, 2M, 5M, 10M, 20M whole marrow. I referred only to single cell transplant studies, because we were discussing the problem of HSC heterogeneity. You can nail it down only on single cell level. Because I don’t study HSC heterogeneity, I don’t do single cell transplants. Practically you sort cells in each well of 96-well plate, control cell presence under the microscope, mix them with 0.2M or fresh competitor cells and transplant IV. Single HSC are functional, do migrate, engraft and repopulate a mouse. You can read the protocols in studies done by groups I mentioned earlier.

        I still don’t understand what do you mean by “Clonal analysis allows us to manipulate a single cell, but serial transplantation does not allow to do it.” As I mentioned many times we can do any thing by single cell transplant and serial transplant. We can also play with single cell in vitro, but possibilities is limited and data should be interpreted with caution.

        About the practicallity of deviding HSC for subpopulations. Frequently it doesn’t really matter for clinicians and medical points of view. Heterogeneity was discovered as biological phenomenon. So, now we know it exists. In clinical HSC transplantation we don’t need to dissect this heterogeneity, because we always transplant enriched HSC/ progenitor cell population or total mononuclear cells. A lot of basic science discoveries have a little clinical significance. For example, why clinician need to know how many defined progenitors exist between common lymphoid progenitor cell and mature NK cell? Maybe 3 maybe 5 maybe 7? Still a lot of scientists continue to work on this and get government fundings. I’m all for clinical relevance.
        The clinical significance of HSC heterogeneity cold come from comparing normal HSC with leukemic SC. If it turns out that leukemic SC are also heterogenous, we should adjust our treatment strategies. For example, the current hypothesis says that leukemic SC are quiescent and to target them we need to make them cycle. But if we extrapolate normal HSC data, we can realize that leukemic SC could switch quiescence to cycling back and forth and become undruggable. The first thing which came up recently about leukemic SC heterogeneity is a great variability or surface markers expression. They can turn them off and on all the time. So, the clinical significance of leukemic SC heterogeneity is that we can’t use antibody to surface markers to target them selectively.

        Finally, about the mice. Genetically identical means the same breed. For example, C57B6J or SJL as recipients. When you do thousand of transplants and you don’t see a difference between all repopulation in normal conditions, you can realize and appreciate how remarkable similar they are and how model is “clean”. Of course, unless you start to titrate cell doses down to single cell. That’s how the whole heterogeneity story unfolded.

        doi:10.3205/maximowaward_2012_blog-comment_11

        Please cite this contribution as follows: Alexey [Bersenev]. Ilya, Colony assay is not assessing stem cells, it’s. Blog comment, Maximow Award contest, May 2012. Cell Ther Transplant/Maximow Award, May 2012;blog-comment_11.
        doi:10.3205/maximowaward_2012_blog-comment_11

  6. Kshama Gupta says:

    Dear Dr. Alexey,

    Many thanks for your comment and making this a very interactive platform. I highly appreciate for your read and for all the valuable information you conveyed. Though not being a clinician, I would agree as you state / with given references that “myeloid biased-HSC” could also lead to multi-lineage engraftment and which infers that there’s no black and white. However, in line with the same, I had tried to highlight that there is yet much needed research going on to decipher the various differentiation routes [the quoted models], that these sub-populations would / may undertake, to bring about such plethora of functions. This further confers that the information we have today, is limited by our ability to really distinguish between these cells cause of the multi-lineage effects they exhibit (under the influence of Microenvironment / stimuli), and hence are heterogenous. Thus, it would be of significance to understand and classify them at the single cell level.
    In agreement with as you said, this may not be very applicable for the patients who must require the whole marrow transplantations but it’s a very relevant aspect for advanced targeted therapies to various kinds of hematological dis-orders. Many leukocytopenia occur by the differentiation block at some or the other progenitor stages as known well also in case of congenital neutropenias (Welte K., Skokowa J. et al.). Therapies currently under effect are quite successful but there still are deleterious clinical observations or out comes as secondary leukemia found in some patients after long term treatments. The exact cause of these may not be the therapy per say but down stream / inherent defects at the molecular level. Some of these are harbored by the specific subset of progenitors. To target the hematopoietic stem–progenitor pool or to perform whole marrow transplantation may not always be a feasible approach and in cases would abrogate the other lineages as well.
    Delineating the individual steps in differentiation cascade v.i.z. when / which progenitor population undergoes proliferative burst, what path it takes, in response to what stimuli and to what counts in normal as compared to diseased has quite lead to our better understanding of the patho-mechanisms involved. Along with, to exploit the behavior and characteristics ( Molecular / functional) of these sub-populations have contributed to several challenging advancements in the regulation/ treatment of various hematological disorders.
    As we all have agreed at some point or the other during our discussion that HSC population is heterogenous, and as quoted by you previously, even in clinics, its this mixed-hematopoietic progenitor cell based therapy which infact furnishes the properties of STEM cells, otherwise; hence we yet need more information to really say which cells exactly are the actual “initial point“ (as said by Ilya) and how do their descendents take their path. Rather than limiting our selves with calling this potential heterogenous population as HSC, with the current knowledge of this pool, a further step in the precise identification and categorization of the progenitor / sub-populations would not only open gates for new and better therapeutic approaches but would help us understand how our complex physiology had evolved.

    Thanking you again for sharing your ideas and for all the valuable inputs / discussion …

    doi:10.3205/maximowaward_2012_blog-comment_15

    Please cite this contribution as follows: Kshama Gupta. Dear Dr. Alexey, Many thanks for your comment and making. Blog comment, Maximow Award contest, June 2012. Cell Ther Transplant/Maximow Award, June 2012;blog-comment_15. doi:10.3205/maximowaward_2012_blog-comment_15

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