This discussion took place at LinkedIn group: International Society on Stem Cell Research in July 2010. This discussion is publicly available.

Do we really need hematopoietic stem cell expansion for clinical use?

 Hematopoiesis.info

For many of you this question may sound strange. A few years ago, like many of you I’d answer - YES, we really need it, no doubt! Recently I’ve changed my opinion and I don’t think we need it. I don’t think we need to spend a lot of time and waste money on searching for the magic “expansion elixir”. I’d be happy to hear any critical comment and to discuss this further. I hope to hear the opinion

 

Alexey Bersenev • David,

My position is that we need to use correct terminology and definitions. When you read CD34+ cells expansion in number (by phenotype) you have to realize that HSCs is only about 3-10% of this population. More of that, so far we don't have any evidence from clinic that we can expand long-term engraftable subset of CD34+ total cells (true HSCs). Instead, we have an evidence that we can expand progenitor cell subsets within CD34+ total population. So, we need double unit cord blood transplant only because doubled number of progenitors within CD34+ total cell population can play significant role in outcome improvement, but NOT because we doubled number of functional long-term HSCs. The problem is that people use CD34+ cell (which contain 90 and more % of progenitors) expansion and HSCs expansion as synonyms.

So my answer is that we don't need to increase number of HSCs any more, because 20 years experience showed that we can not do it, but we need to work on progenitor expansion instead and think how to increase homing and engraftment efficiency of fresh HSCs in single BM or CB unit.

I'd be happy to see any link to any work about the efficient HSCs expansion in clinical settings.

 

Robert Oostendorp • Is there any evidence that we even can expand stem cells? Some claim it can be done. However, using the most stringent definitions of the functional abilities of a stem cell, ex vivo expansion has only been achieved two- to four-fold. And even in such studies, there were no stringent controls enabling quantitation of the extent of self-renewal. I am aware that many scientist simply simplify the definition of human hematopoietic stem cells to "CD34+ cells," but, it has been shown by many investigators that in expansion culture CD34 as a single marker is not reliably quantifying long-term repopulating self-renewing cells.

Whether we need stem cell expansion? No, for most clinical applications we don't. Unless we want to use single cord blood units to treat adult patients, or perform gene therapy on single units. As long as we do not understand what makes a stem cell self-renew, attempts at mimicking this process ex-vivo will be difficult.

 

Alexey Bersenev • Exactly my point Robert!

 

Alexey Bersenev • Robert,

What we know about self-renewal of adult stem cells so far is that almost complete overlap with "cancer program". If you look at all of pathways that you know today. So if we want to stimulate self-renewal ex vivo in order to expand stem cells, we have to realize that we're launching "cancer activation program".

Nobody knows where the "safety border" of mutation-free expansion, it's too fuzzy.

 

Dr.M. Chandrashekhar MD • How about Mesenchymal Stem Cell isolation ? How do we do it from Umbilical Cord/ Bone Marrow derived Stem Cells ?

Iam a curious , ethical ,arm chair stem cell enthusiast with no lab experience !

 

Sherri Schultz • Is HSC population purity relevant if the expansion technique used over the past 20 years does not yield a homogeneous population, but is successful? Maybe that degree of heterogeneity IS part of the equation for success in this situation.

We need stem cell expansion if we want to have the possibility of multiple new application options in the future. We can currently expand and maintain pure populations of pluripotent embryonic stem cells as well as multipotent adult stem cells isolated from different organ origins. More research will bring solutions to HSC and other stem cell expansion. Have patience, we are a young field!

 

Philippe Guardiola • I think it clearly depends on what you want to do with your cells. For allogeneic transplantation of hematopoietic progenitor/stem cells (HPCs), using bone marrow and more frequently now GCSF mobilized peripheral blood stem cells, as we call these, we probably dont need any expansion. For autologous transplantation of HPCs, it has been done in the past and this was not really relevant with limited clinical benefit. This approach became obsolete with the arrival of GCSF and mobilization of HPC. Now for cord blood transplantation, especially in adults: pooling 2 CB units might be the choice but are we sure that it is because the number of infused HSC is higher than it is why these grafts work maybe better ? The interesting thing here is that when we used for transplantation a single CB unit following a fully myeloablative conditionning regimen the time to achieve neutrophil recovery was about 4 to 6 weeks. Now with reduced intensity conditioning regimen + GCSF it is beow 3 weeks in most cases... cytokine storm behind this ? So here it is even not that clear for me. Finally regarding expansion of other stem cells, for mesenchymal stem cells there might be some concerns here about the acquisition of cytogenetic abnormalities...A french trial was stopped because of this...so maybe not that easy. For the other cell types I cannot say.

 

Sherri Schultz • Philippe, it would be interesting to hear about the study parameters of the above mentioned treatment (N=?, disease status, etc). Is this information published?

By the way, any group of cultured cells can acquire cytogenetic abnormalities. Purely by the nature of passaging cells multiple times. It is the responsibility of that laboratory to monitor the health of their cultures in order to accurately report results based on the karyotype they claim - for the sake of our current knowledge base and I guess, especially for the sake of having a safe clinical trial.

 

Robert Oostendorp • To Dr. M. and Philippe: Regarding mesenchymal stem cells, I'm not sure these cells exist. If the definition is that a single stem cell should be able to regenerate a tissue and self-renew, this has only been shown for hematopoietic stem cells. Like with blood cells , it is easy to make mesenchymal stromal cells (MSC) grow. But, also like with blood cells, when you do so, you lose multipotency very quickly. The problem with MSC is that we do not know what the stem cell looks like, so we have no way of quantifying the success-rate of expansion. The same question applies, though: is MSC expansion a clinical necessity? Most current studies on therapies proposing to use MSC could be implemented perfectly fine when only mesenchymal progenitors (mostly osteoprogenitors) were expanded.

 

Alexey Bersenev • @Sherri Schultz -

HSCs population purity is irrelevant expansion techniques, unfortunately. This is the main problem that I see now. Today we have evidence only for progenitor cells expansion within CD34+ but not stem cells.

The best way to assess this before clinical trial could be following:

take 1 CB (or BM) sample, split equally in terms of total MNC number or CD34+ total sorted cells; 1/2 transplant in NSG mice fresh before expansion, 1/2 start to expand for 2-4 weeks (you can make same input by CD34+/CD38- or more pure HSCs - CD34+/CD38-/CD90+/CD45RA-) and transplant after that in NSG mice; then you start to analyze output - blood lineages chimerism and human+ cells in the blood and BM in primary transplant, secondary and 3rd transplant. After this you can draw conclusion about your HSCs expansion efficiency.

Even nobody done this yet pre-clinically, we've got clinical evidence already that we have nearly zero expanded HSCs in CB sample (Bernstein trial in Fred Hutch). The clinical test could be keep track of chimerism-engraftment in the patient in few years after transplant and show that expanded cells able to repopulate blood lineages in long-term.

Now we know pretty much all stem and progenitor populations withing CD34+, now we can assess them. So we should use correct terminology and talk about expansion of progenitors but not stem cells. It's our time to make clinicians and scientist to think in different way and move on.

When you talking about 20 years of success in HSCs expansion what work do you keep in mind? I'm keeping track of all published studies and didn't see any indication to successful HSCs expansion in clinical settings. There were some trials 5-8 years ago, I indicated here - http://bit.ly/d7MrWgBut all of them assess total cell number expansion and clinical necessity, but not HSCs number and function. They even didn't talk about stem cells. By number of reasons these trials didn't progress to phase III and didn't show great clinical improvement and Philippe mentioned above.

 

Alexey Bersenev • @Philippe -

2x unit CB transplant still a black box, I think. Even we can see the benefit of this approach in many indications, we don't understand how it works. Theoretically you would expect a doubled number of HSCs and progenitors and expect everything doubled clinically, but practically we see mostly one unit HSCs engraftment out of 2, and sometimes we don't see improvement in neutrophil recovery (last trial from Wagner group - http://bloodjournal.hematologylibrary.org/cgi/content/full/114/19/4293 ).

So seem like we can get clinical improvement in some indications by combining 2 CB samples versus 1 unit transplant, but it could not be explained by doubling HSCs and progenitor numbers. We don't understand mechanisms yet.

 

Philippe Guardiola • @Alexey - that s exactly the point. Only one of the 2 CBU will survive in most cases and there is not any clear factor that can predict which one will stay and which one will disappear to my knowledge. But one hypothesis could be that, best results might be related to the fact that one CBU is "dying", inducing apoptotic bodies and immune deregulation such as "a tolerance state" maybe helping the other CBU to get implanted more efficiently... this is just an hypothesis...

 

Robert Oostendorp • It seems the rift between basic scientists and people working in applied science is growing. In the human system, there is no reliable phenotype of stem cells from "expansion cultures" the only test we have of expansion we have is the limiting dilution assay in NOD/SCID (or other) mice. The number of groups that have reported such experiments can be counted on the fingers of one single hand. And, from a basic scientist point of view, there is no experience with HSC expansion, since not one single group has reported reliable and reproducible, clinically applicable increases in the number of repopulating self-renewing HSC.

 

Robert Oostendorp • To Philippe, I noticed in my cultures with cord blood, that culturing different donors together adds about twice the number of progenitors. Not sure about the HSC, since we didn't do NOD/SCIDs on those. So I think the two donors interact and maybe synergize with eachother. Both donors survive in culture, but at some point, one of them starts to dominate. We didn't notce over cell death in just one of the donors, so I'm not sure, but the apoptotic body hypothesis coupled to TLR signaling may be involved, as you say. The real mechanism? I guess there's more capable groups than mine working on that, but I believe the multiple donor strategy may indeed be a breakthrough. In that case, the riddle of expansion does not need to be solved at all, just add more donors to the mix.

 

Steve Wolpe • There's good evidence from the Seattle group and MSKCC that dominance is due to multiple factors. The two main ones are viability (many times one cord is not fully viable) and immunologic (T cells in one cord reject the other). The issue about expansion also is two-fold. Clearly progenitors can be expanded and give rise to accelerated recover of neutrophils. The question is whether HSC's can be expanded and whether this is necessary. Is it suffficient to have more progenitors and more rapid neutrophil recovery and allow for endogenous stem cells to reconstitute or do you need donor-derived long-term HSC?

 

Alexey Bersenev • @Dr.M. Chandrashekhar,

I'd agree with Robert at the point of MSC expansion. First of all MSC is poorly characterized in order to call them "stem cells". Many people in the field prefer to call them "mesenchymal progenitors". Second, at many indications we don't need pure expanded MSC, but we need their derivatives - osteoprogenitors or chondroprogenitors. The value of expanded non-differentiated MSC in some "non-skeletal tissue diseases" still debatable and under investigations in ongoing clinical trials.

 

Alexey Bersenev • I'd like to share my collection of bookmarks on "2x cord blood transplant" -

http://www.connotea.org/user/nanog/tag/2x%20cord%20blood%20transplantsome studies started to unveil mechanisms of cells interaction in both samples and preferential engraftment just one.

I think, other interesting approach is split doses transplant, when 1 or 2 samples of CB or BM can be divided for few doses and transplanted in 2-5 procedures/ days. The recent clinical trial results - http://www.ncbi.nlm.nih.gov/pubmed/20569092The rational behind of this approach is limitations of BM niches saturation per day and their "re-cycling". It was proposed in experimental work by Irv Weissman group - http://jem.rupress.org/content/206/12/2837.abstractWe don't know of the relevance of this model to conditional regimen in clinic, but it gives us some clue. Conditioning become less and less toxic and there is a trend from myeloablative to minimal (reduced) non-ablative regimen. So, I don't think that conditioning kills a lot of true quiescent HSCs and destroying niches. In this case niche-saturation hypothesis should work. Whatever huge number of HSCs you inject, only limited niches /per day available in BM for their lodging and engraftment, all other HSCs go to waste. But if you split doses and will keep transplanting HSCs daily, you will increase engraftment and improve transplant outcome. Again, we don't need expansion here.

 

Weiwen Deng • A possible approach to expand HSCs is to "expand MSCs first, then reprogram these cells to iPS cells, then HSCs". Because the real stem cell in bone marrow is probably mesenchymal stem cell (MSC). This is like the situation in brain: astrocytes first, then neural stem cells, and neurons last. The percentage of MSCs in bone marrow is 0.01%. The percentage of HSCs in bone marrow is 1% or more. So it makes sense that MSC is the earlier stem cell than HSC in bone marrow.

 

Dr.M. Chandrashekhar MD • Thank You, Robert & Alexey. Point taken.

 

Igor Katkov • ha-ha-ha, bravo Weiwen, it's like scratching one's left ear with one's right toe ;-)))

The question, rised by Alexey: do we really need to expand HSCs???

 

Daria Neidre • Thanks for this discussion. Something related to this came up in my proposal defense. As we are looking at mainly MSCs from various tissue sources, but there was a comment about the potential of HSCs and their role in the clinical setting when implanting, specifically for orthopaedic procedures. Our lab does not focus on HSCs so we were unable to truly quantify or address that topic. I'll keep following this discussion to see if there is a way we can answer the clinical question of the role of HSC in a clinical implantation environment. Thanks!

 

Aasawari Bapat • @Alexey ...........thank you for the great link..........very useful........

 

Jim Hardy • We should try to get someone from Lonza to comment. They most certainly expand MSC in vitro for the Prochymal product and for the clinical trial material (all expanded MSCs) for Athersys.

 

Weiwen Deng • I have already built a MSC bank from 48 healthy bone marrow donors. I can readily expand the cells 1000 fold in 14 days. I believe that MSC will be the first adult stem cells for clinical applications in a couple of years.

Philippe Guardiola • Have you been studying cytogenetic, transcriptome and methylome for instance with microarrays to see if there were some modifications betwenn t0 and end of the expansion ?

 

Weiwen Deng • I have done cytogenetic assay and my SKY data shows there is no chromosome abnormality after ex vivo expansion of human MSCs. However, there are extensive cytogenetic abnormalities after only a couple of passages of mouse MSCs. This is obvious one difference between human and mouse MSC.

As I do not have big funding, I cannot afford transcritome and methylome assays. I'll be happy to do microassay if I can get a grant to do so.

 

Philippe Guardiola • How do you explain, Weiwen that in mouse you have cytogenetic abnormalities and not in human, the more so that in mouse you have longer telomeres - if I m not wrong - ? Have you been doing high-density SNP-arrays or CGHa to detect small abnormalities microdeletions/insertions and/or LOH ? That would be interesting to get these kind of data

 

Alexey Bersenev • Philippe and Weiwen,

you may be interested in reading my recent post where I analyzed new data about spontaneous transformation of expanded human MSC -

http://bit.ly/cbUWN8People done STR and DNA fingerprinting, I can attach/send last 2 publications about it.

Alexey Bersenev • If I'd be a physician, injecting expanded HSCs or MSCs into my patients, number of cells with chromosomal abnormalities/ per million in cell product will be not informative to me. I pretty much don't care where these abnormal cells came from - spontaneously transformed or cross-contaminated culture from cancer cell lines. But I'd be really really care to know how many cells with chromasomal abnormalities is enough for clinical onset of malignancy in my patients in months or years after injection.

It's unknown, it's could be very fuzzy - maybe 10 cells for me, but 50 cells for you? Where is the border? How many injected abnormal cells immune system can eliminate safely and efficiently?

These kind of things are not discussed in cell therapy community yet. Very unfortunately. That's what Prockop started to talk about in his editorial

http://www.nature.com/mt/journal/v18/n7/full/mt201099a.html 

Weiwen Deng • As human immune system can kill 100,000 spontaneously transformed tumor cells per day in our body, we do not need to worry about the number of transformed MSC Prockop talked about.

 

Philippe Guardiola • I would say that the problem is more about acquiring a specific oncogenic even... Plus if I believe what is published, these cells have a clear immunosuppressive effect as some have been used to treat graft versus host disease (correct me if I m wrong I m not working on this topic)... so maybe allowing the growth or few residual malignant cells... explaining what was called tumor contamination by some ?

 

Jim Hardy • Oops. I just realized that Alexey's *real* question was expansion of HSC (not MSC) and I inadvertantly changed the topic based on @Roberts comment about expanding any stem cell. I do not believe anyone hads been able to demonstrate expansion of HSC to this point in time. Does anyone know labs that are still working on expanding HSC from Bone marrow or Cord blood/placental tissue?

 

Weiwen Deng • Nobody can expand HSC!!! This opinion has been repeatedly emphasized by Dr. Irving Weissman who has spent 4 decades trying to do so. If we can figure out a way to reprogram MSC to HSC, we solve this problem. Because we absolutely can expand MSC.

 

Steve Wolpe • It's very well known that human cells are more resistant than mouse cells to develop chromosomal abnormalities in culture. This is not an MSC-specific effect but applies to a wide variety of cell types. Mouse cells quickly transform in culture whereas human cells are more resistant to transformation with passage. That said, there is always the danger that a minority of multiply passaged human cells will transform. Current assays are limited in their ability to detect rare occurrences like that. A single transformed cell can cause cancer as demonstrated by the fact that most cancers are clonal - i.e., they are derived from a single cell.

 

Igor Katkov • "Nobody can expand HSCs"! Than what is this all discussion about??? I envy you guys, you have time to talk for days about a topic NOBODY can do!

In regards to reprogramming MSCs to HSCs, it is easier and more logical to reprogram ANY cell to iPSCs and then differentiate to HSCs (though I know it'll be not easy). As I said several times, Mother Nature doesn't like shortcuts: you have to clean you chalkboard (epigenetic profile) to the level of young immature embryonic-stem cells and only THEN to creat smth new. The right ear is not growing from from the left toe, unless somedy's mother was sitting near an atomic reactor for 10 years ;-)

 

 

Philippe Guardiola • Back to initial question: do we need expansion of HSC ? What for, I would ask first. As an hematologist my answer is currently NO ! Even for cord blood transplantation we can pool 2 or 3 of these to achieve the cell number we want. Then after, there are so many researchers working on things they are interested in but have no meaning (and might never have any in the future) or application for clinics...

 

Alexey Bersenev • Philippe,

completely agree on HSCs expansion for clinical needs.

 

Philippe Guardiola • But deriving to MSC, in this discussion, is of interest for me anyway ! ... even if I dont think "today" that these cells are so useful for clinics !!! The enthousiasm there was few years ago for the treatment of GvHD is not so high nowadays... fashion victims ?

 

Alexey Bersenev • Regarding MSCs for GVHD - clinical trials still going on. So we don't have definitive answer, but interest still persist. My rough guess is about 10 of them currently on phases I-III around the world. I'm trying to track them all.

Osiris got to the point of phase III multicentral, including Canada. Waiting some reports by the end of this year.

 

Igor Katkov • Here is a list of GVHD clinical trials:

http://www.clinicaltrial.gov/ct2/results?term=Graft-versus-host+diseaseit refefers 655 clinical trials. Which numbers are MSCs?

 

Igor Katkov • I found 19 more or less relevant:

http://www.clinicaltrial.gov/ct2/results?term=Graft-versus-host+disease&recr=&rslt=&type=&cond=&intr=mesenchymal+stem+cells&outc=&lead=&spons=&id=&state1=&cntry1=&state2=&cntry2=&state3=&cntry3=&locn=&gndr=&rcv_s=&rcv_e=&lup_s=&lup_e=I would narrow them to 12 relevant, so 12/655 = 1 of 55 < 2%!

 

Philippe Guardiola • One thing about MSC and GvHD treatment that I have understood (I think) is that if you stop infusing these cells the reaction reappears and is even worse than it used to be at the beginning.

It is good to know that there are still some trials with these cells in this context; we need to learn more about these cells and maybe use them differently...who knows.

It is also clear that medications to treat this reaction are and will be easier to use than any kind of "prepared" / modified cells as of today, for the huge majority of transplant centers.

We have to keep in mind that the main problem with the treatment of GvHD (at least acute GvHD) is not to stop this reaction but to avoid collateral side effects such as infections because of the immune deficiency we induce with the drug used to treat GvHD. More patients die from infections such as viral or fungal infections than from the direct consequences of GvHD in this setting.

 

Robert Oostendorp • @Weiwen: the failure of one person to expand HSCs does not mean that it's not possible.

@Igor: I can assure that HSC expansion takes place in vivo, especially in the embryo.

The fact that researchers cannot repeat this trick only means our lack of understanding. But, do we need to expand them? For me, HSC expansion is a scientific challenge, a puzzle so to speak. I'm convinced that puzzle will be solved.

With regard to MSC. Personally, the problem which applies to HSC, I think also applies to MSC. I do not believe currently there are known culture conditions which allow expansion of true multipotent bone-forming, hematopoietic-environment-recruiting MSC either. As I argued earlier, the fact that cells proliferate is not proof of stem cell expansion. Rather, in the hematopoioetic system, rapid proliferation is usually associated with loss of stem cells (perhaps not when you also express HoxB4 or the like).

Infusing MSC is like infusing magic. We have no idea what we are infusing, nor what the cells we infuse are capable of. As a basic scientist, I would be extremely cautious with such cells.

 

Philippe Guardiola • I totally agree to all your comments Robert !

HSC has been and is still a good model.

Playing with fire is what we have been doing for decades with allogeneic bone marrow transplantation... and still doing...but for sure, regulations are not the same as they were 30 years ago.

As Pr Rainer Storb said, if we were to start allogeneic SCT nowadays, what we have been doing in the past at the beginning of that challenge that would be impossible because of all the current regulations...

So, even if I m not convinced by the first results of MSC therapy for GvHD it is worthwhile continuing clinical trials with these. Gene therapy for SCID children had also a huge side effect with these induced leukemias but we have learnt from this... Same will apply to MSC if regulations and indications of these cells are adapted to the risk the patient is facing.

Cell therapy is not that easy because the product is not clearly characterized in most cases...we faced a similar problem with dendritic cell infusion programs few years ago by the way... Most of these programs have stopped.

 

Ross La Motte-Mohs • There is no evidence that MSCs are the "real" stem cell in the bone marrow. They have yet to be purified to homogeneity (they've almost purified mouse HSCs to homogeneity, human HSCs still require further characterization), lack clear identification markers, and thus far do not give rise to HSCs. If fact MSCs give rise to separate lineages that do not coincide with HSCs save that both these cells are found in the bone marrow. MSCs are multipotent (i.e.: bone and stromal elements) just like HSCs are multipotent (i.e.: white and red blood lineages). Since both are mesodermally derived, it is more likely that at some developmental stage, there was a bifurbication from a progenitor cell that can give rise to both MSCs and HSCs. As of yet, this progenitor has not been isolated in vivo within adults. It is conceivable that such a population exists in the bone marrow and is minor population of the heterogeneous MSC or HSC population. Percentages in the bone marrow of heterogeneous populations tell you nothing about function or differentiation potential. One needs to isolate a relatively pure population of the candidate cell population (preferably at the single cell level) and test their differentiation potential in vitro and in vivo in LDA and competitive repopulation assays to address that question.

It is conceivable that you could generate an iPS cells from an MSC and then drive it back towards and HSC fate. However, generating of iPS cells is grossly inefficient process, and questions remain about the safety of transgenic resetting of the developmental program (propensity to cause cancer, epigenetic differences, issues with gene regulation/disruption). This doesn't mean that it is impossible, rather it may not be feasible in the clinic.

Finally, the general consensus is that more of the “right” cell is advantageous to improving stem cell engraftment. That being said, in theory all you need is one stem cell to engraft, but in practice additional cells (stromal or progenitors) that support stromal regeneration may be needed to supply the niche (which is damaged during myleoablation) to accept stem cells. Expansion technologies may expand HSCs there are examples out there in the literature, but you also have to take into account of that the definition of a stem cell must extend beyond the phenotypic characterization and demonstrate function engraftment of all subordinate lineages. The vast majority of technologies are expanding the progenitors found within the CD34+ fraction. This is because the definition of a stem cell remains ambiguous. It should be noted that the source of “stem cells” and “progenitor cells” varies in engraftment ability as cord blood HSCs are not entirely the same as bone marrow HSCs, nor the same as mobilized CD34+ PBLs, nor are HSCs derived from ESCs or iPS cells.

 

Steve Wolpe • Pooling 2-3 cord bloods is very expensive ($35K.cord) and current data don't show that you get any real benefit in terms of engraftment. Almost always, one cord takes over and the other does not engraft. HSC expansion is needed in cases where cells are limiting (e.g, cord blood, poor mobilizers, etc). The question is whether you need expansion of the stem cells or just the progenitors. The idea behind the latter is that they give a burst of neutrophils to fight infection until the HSC's can engraft and regenerate. The real question is whether you need expansion of long-term repop cells, which so far has been difficult to achieve.

 

Alexey Bersenev • @Steve,

1. Expansion of long-term repopulating human HSCs (this is equal HSCs definition, all other cases are not HSCs) has never been achieved so far. Correct me with a link if i'm wrong.

2. 2x CB unit transplant we intensively discussed above (page 1) and this is exact the case which demonstrates that we don't need expansion of CB for clinical benefit. 2x unit CB works by some magic reasons, but not because you increase HSCs number or engraftment. This is exactly my point.

3. We can compare the cost- price - benefit ratios if both products on the market. Do you know how much cell manufacuring going to cost for HSCs expansion protocol? 2-4 weeks of culture, labor, GMP-facility support and so on. Probably still cheaper than purchase 2nd CB unit, but so far it's just a speculation. Tell me the price for expanded HSCs service available on the market and I'll tell you which procedure is more expensive. So far, we just got only 1 HSCs expansion trial on phase I, which showed that HSCs expansion is impossible (Bernstein at Fred Hutch). I think all of companies, which running trials and propose HSCs expansion should honestly re-name it to "progenitor expansion".

Also I think price for 2x unit CB will depend on CB bank - hospital relationships, country and regulations.

4. As Philippe mentioned above and I agree with him, in almost all indications in hematological clinic we can cover necessity of HSCs expansion with something else, which works good. Transplant physicians do CB only if they can't find matched BM or MB or patient is poor mobilizer. In case of CB you have options to choose a sample with good MNC or total CD34+ number or 2x unit transplant.

 

Alexey Bersenev • @Ross,

I think you made good points.

Only thing I'd correct is that we know now how to dissect all HSCs/progenitors subsets in human. You can read here -

http://bit.ly/9i2rgP

http://bit.ly/cbjdpVSo now, based on this knowledge somebody should take a hard work and really test the possibility of expansion each of mentioned HSCs/progenitors subsets (from the same sample of CB) side by side in LDA, competitive repopulation, serial transplant with control of fresh cells.

 

Steve Wolpe • I think you're caught in a Catch-22. Transplant physicians currently only do CB when they can't find matched donors but that's because there isn't a good expansion technique. CB has a number of advantages over BM or mobilized PB but they aren't realized because there aren't enough cells in CB to transplant an adult. What's needed is a good expansion technology to make CB in adults a useful technique. The Bernstein results didn't show that "HSC's expansion is impossible"; they just showed that notch ligand is not sufficient. Indeed, as one correspondent noted, HSC expansion happens in vivo so it is entirely possible. We just don't know all the factors necessary. Harvey Lodish published a while back that he got HSC expansion using angiopoietin. Others claim that they have the special factor or factors necessary. Gamida Cell is in an international Phase III using a copper chelate and Mesoblast recently announced that it is going into PhIII as well based on excellent PhII data. I know of some major pharma that also have programs about to go into the clinic. Evidently they've done the cost/benefit analysis and concluded that expansion is worth the effort.

 

Alexey Bersenev • Steve,

You're right about Bernstein trial, I should not use "impossible", I should say "HSCs expansion was not achieved". My mistake.

Agree, CB has advantages and BMT guys know them, so I think they are realized it, because some data is out and published. But why wouldn't we say transplant physicians use CB as a 3rd option only because they don't know how to increase HSCs engraftment rate. I'd also argue, that we probably have good enough HSCs number in one fresh average CB sample for average adult recipient for long-term repopulation in months and years, just make them home better and make them engraft better. I'm sure, these kind of approaches will be much cheaper than a month culture in GMP conditions.

Also with stem cells expansion we always have to keep in mind the following: any game with self-renewal is very dangerous game. You activate self-renewal program ex vivo = you launch carcinogenesis.

As far as I know all companies that you mentioned have no any solid experimental or preclinical evidence that they expand true engraftable long-term HSCs, phenotypically Lin-/CD34+/CD38-/CD90+/CD45RA- tested in serial xenotransplant with freshly sorted HSCs in comparison. I may be wrong, but again, give us a link.

The fact that they claim increasing CD34+ total or even CD34+/CD38- cell number does not correlate with HSCs engraftment.

According cost/benefit analysis, yes they sure that it worth efforts, but they should read more (this discussion at least) to realize that it is progenitor cell expansion but not HSCs. As I indicated in my post - I'm proponent of progenitor cell expansion. These guys do all clinical magic first 1-2 months. These guys easy to expand and we should use right terminology. So I'm sure that progenitor cell expansion is relatively cheaper than 2x unit CB, going to be cost effective and absolutely worth our efforts.

 

Igor Katkov • TO ALL,

Do you actually know how much valuable HSCs are lost or "corrupted" during the cryopreservation process? Beside some poorly designed exps, no one really has investigated that matter. From my own experience, SCs are in fact much more senstive to the stresses related to all steps of CP procedures than progenitors and differntiated cells, and it has a clear explanation. They, for example, can be easily killed or forced to differntiation by over-centrifugation, and, at the same time, can be easily lost in supernatant if centrifugation time/force was not enough to spin them all down. Just an example...

http://www.ncbi.nlm.nih.gov/pubmed/10736749 BTW, that sedimentation technique for CB really sucks. And this just a beginning.

So, my advise is that instead (or beside) of pouring big $$ into HSC expansion or combining two doses, etc, BMT folks should invest a fraction of it and hire PROFESSIONAL cryobiologits, not self-educated amatures, for that kind or research. May be increasing the yield of VIABLE and FULLY FUNCTIONING cells on 30-40% would solve a lot of issues that you hav been talking here about?

 

Philippe Guardiola • What are the aims of doing an allogeneic transplant ?

1- hematopoietic recovery is efficient especially for neutrophil recovery that we d like to obtain between day14 and day21 ideally ... not too fast because usually we get by the same time severe acute GvHD... at least in the current way we do SCT

2- the graft engrafts which can be different from point 1

3- the disease is controlled and there is no relapse

here, we know that the graft versus disease effect is or can be pretty important (remember T cell depletion to avoid GvHD and the high rate of relapse that was observed following this kind of procedure)

4- the procedure itself is not killing the patient because of GvHD, infections, toxicity mainly

Currently with CBT you get

- faster hematopoietic recovery than in the past for many patients but not all (at least for neutrophils) and it is not so predictable even if the cell dose is important as well as the intensity of the conditioning regimen. Keep in mind that nowadays most of these patients received GCSF from day0 until neutrophil recovery is fine, which was not the case in the 90ies early 2000s.

- we still have more failure of engraftment than with marrow or PBPC for sure and this can be lethal pretty fast

- much more complications, especially viral infections so that the price of the hospital stay is much higher than with PBPC, at least for the initial in-patient unit stay (survival curves are the same as with other stem cell sources but the real life during the first hospitalization is clearly not the same...)

- we get less acute GvHD but for chronic GvHD it is not so clear even if it could be the same

- the immaturity of the CB cells makes me wonder whether the graft versus disease effect, mediated by some kind of lymphocytes, is as efficient as it is with PBPC... not that sure and that s an important point that will be clarified during the next decade.

If you can get a faster neutrophil recovery with 2 CBT instead of one and the hospital stay is shorter by let say 10 days (remember with one CBT unit it can last between 10 days and 60 days, median around 21-30 days) then it is 10 x 1500-2000 $ + price of the anti fungal drugs and other antibiotics > 10 x 1000$ then the second CBT unit is not that expensive... and I would recommend not to buy those CB units in certain places where it is much more expensive for reasons I dont understand / believe

Remember that one of the major interest in CBT is that it tolerates HLA mismatches so that you can find a donor pretty quickly; the more so that it is always available in less than 2 weeks... So for patients who urgently need a SCT this is a major point... what if you have to do expansion...?

The interesting thing would be to identify predictors of slow hematopoietic recovery with high specificity and sensitivity. Then for those we could think about progenitors expansion if we have time to do this... but here again it will be more complicated to perform than to pool 2 CB units if this later procedure is also effective to fasten hematopoietic recovery see the recent paper from the Hutch on dogs...

A Preclinical Model of Double- versus Single-Unit Unrelated Cord Blood Transplantation.

Georges GE, Lesnikov V, Baran SW, Aragon A, Lesnikova M, Jordan R, Laura Yang YJ, Yunusov MY, Zellmer E, Heimfeld S, Venkataraman GM, Harkey MA, Graves SS, Storb R, Storer BE, Nash RA.

Prep of the unit is probably something that could be improved also

Finally as 007 says: never say never... for HSC expansion !!!!

 

Weiwen Deng • To expand HSC, we need to know how CD34+ cells were recognized by Dr. Irving Weissman some 30 years ago? I can recall he developed an antibody to CD34 on mouse bone marrow cells and it worked. Then he developed another anti-CD34 to human bone marrow cells. Can anybody share the detailed information of Irving's Nobel-prize-potential discovery? Thanks.

 

Philippe Guardiola • Correct me if I m wrong but HSC are supposed to be CD34 negative and probably PROM1 negative as well (I m thinking about old paper from the same I.W. on mouse hematopoiesis) ?

 

Alexey Bersenev • Human HSCs = Lin-/CD34+/CD38-/CD90+/CD45RA-

Mouse HSCs = Lin-/Sca1+/cKit+/CD34-/Flk2-/CD150+/CD48-

 

Steve Wolpe • The most recent data suggest that a double cord blood does not accelerate engraftment over the use of a single cord. In almost all cases, it's only one cord that engrafts. Instead, double cord blood grafts increase the chance of getting a good, viable cord. See the recent papers by Mary Laughlin, who first developed the double cord blood technique. The negative aspects of CBT that you cite are due to the low dose of cells that are obtained. When used in children, CBT is superior to BM or mPB and is the method of choice nowadays. If there were a way to expand cord blood numbers it would likely be the method of choice in adults as well given the advantages in terms of reduced GVH and equivalent effects in relapse rates (see the recent study in Lancet).

 

Igor Katkov • Nice, Alexey!

I am not surprized some folks here, who have been discussing this issue for weeks, don't know the difference between human and mouse HSCs!

 

Igor Katkov • Steve,

It just enforces my point that more efforts should be put on the processing and banking efficiency. In contrast to a 2x from diferent donors, if one increases viable yiled from 25-40% (which is REAL yield now) to 60-70%, it should accelerate engrafment because it's the same sample's CB.

But it looks like I am speakimng to a wall, I am outta here!

 

Jim Hardy • Igor, I've been trying to get to people to listen to the same thing. So what I am doing, instead of cannulating and "milking" cord blood is to perfuse the entire placenta. We can get 3X and I think 5X more usable HSC cells doing this than "standard" cord blood banking techniques. Hariri is doing similar work at Celgene's LifeBank USA facility, but he still collects the cord blood first, then perfuses the "exsanguinated" placenta.

If anyone would like to work with me on this (I am trying to find a viable clinical partner), please let me know.

 

Igor Katkov • Jim,

Good idea! Now, multiply your harvest x my double yeild after freezing, we'll get 6-9X of the "standard" protocol!

I've heard one company in Canada banks the whole placenta!

 

Philippe Guardiola • Dear all some of you should not be so full of certitudes. The litterature about CD34- human HSC is clearly not zero at all, and given the fact that this marker is epigenetically controlled and can move from positive to negative and vice versa makes me very cautious about the fact that human HSC are "only" CD34+ (indeed they are certainly CD34-)...Dont forget that we are limited by the technology here... Cheers

 

Alexey Bersenev • Igor and Jim,

completely agree with you - we should re-direct our efforts to increase yield of total MNC and CD34+ from one CB unit (even if its mixed with placental blood) by improve processing and cryo- and banking, instead of asking for billions $$$ for continue develop HSCs expansion techniques.

There is nothing better than fresh cells (to get the same engraftment-chimerism from cultured cells compare to fresh you have to multiply their # 2-10 times) and if there is any way to get than much more that we have now in one unit, we should go for it!

 

Philippe Guardiola • Jim have you contacted Eurocord clinicians ? Either Pr Gluckman or Dr Rocha both are on linkedin.

 

Alexey Bersenev • @Philippe,

You're right there is a big fraction of human HSCs in CD34- population, my rough guess 10-40% of all human HSCs, we're missing them when do CD34+ positive selection. There is some literature about human CD34- HSCs. The problem with CD34- is that we can not dissect them phenotypically - how I showed for CD34+, so we can not study how potent they are rigorously.

So the best case in processing could be Lineage-negative total population which can cover all (CD34+ and CD34-) HSCs. That's why clinicians frequently rely on total MNC number in the sample or on results of colony assay.

 

Philippe Guardiola • Cd34 expression on HSC as many other markers are not black and white - just consider dendritic cells and the way they have been defined along the past 2 decades... same apply probably for lymphocytes... nothing is fixed, everything moves as a dynamic process. It is just that we are not clever enough to manage this continum. Human brains prefer 1 / 0 signal rather than a continuous one, which is in most situation the reality...we are between 0 and 1 not 1 or 0 except when dead !

 

Ross La Motte-Mohs • @ Alexey

I agree, I'm well aware of these phenotypes, but when you look in the clinic, HSC transplants are not using these panel of markers (perhaps they should be – do a clinical trial), they are using CD34+ cells and/or T depleted cells. This is because higher CD34+ numbers increase engraftment. This has been well-established clinically, so if it works you don't change the system. (That doesn't mean you shouldn't try). However, if you want to change the clinical methodology, one does a clinical trial and establishes a better therapeutic. This is a difficult step/leap to put into practice. Clinical trials are slower to accept what's published in the literature not because they are ignorant, but because you have to show a measurable improvement. So while the purist in me says we probably don’t need HSC expansion if you satisfy the mantra “one perfect stem cell inserted into a rejuvenated/supportive niche yields complete, efficient and rapid engraftment,” the practical side says we’ve yet to achieve that goal and we should still be pursuing HSC and progenitor cell expansion.

First, rather than join the Irv Weissman Fan Club, (I admire his work too), I’ll circumscribe my comments to the data. To be blunt, some of the statements by Weiwen Deng are over-zealous, incorrect and not supported by the current literature.

Second, and I agree with you Alexey, the precise phenotypes are indicative of highly purified populations of stem with progenitor cells, but they are not absolute (in humans or in mice). There is near homogenity with the mouse phenotype (SLAM phenotype).

Regarding mouse HSCs = Lin-/Sca1+/cKit+/CD34-/Flk2-/CD150+/CD48-, I don't doubt the phenotype nor really the functionaly outcome -- yes there is long term immune-reconstituting potential. I'm saying the functional test of this outcome is nearly there. Meaning 1 stem cell reconstitutes efficiently all blood lineages (key word efficiently). The discussion of the SLAM paper makes that point. Mouse HSCs have been purified to near homogeneity but not ABSOLUTE, which would be the "true" definition of a hematopoietic stem cell.

Regarding human HSCs = Lin-/CD34+/CD38-/CD90+/CD45RA- again this phenotype isn't in question. The functional outcome is relatively convincing, but more experimental evidence is required. The question is how best to test this convincingly? Immunodeficient mouse models (NSG mice) support human engraftment but it will be difficult to show in vivo within this model system that one cell efficiently engrafts all lineages. The engraftment potential is only as good as the system, and for the moment the NSG mouse is the best system we currently have to test in animal models.

Regarding human HSCs = Lin-/CD34+/CD38-/CD90+/CD45RA- again this phenotype isn't in question. The functional outcome is realitively convincing, but more experimental evidence is required. The question is how best to test this convincingly? Immunodeficient mouse models (NSG mice) support human engraftment but it will be difficult to show in vivo in this model system that one cell efficiently engrafts all lineages. The engraftment potential is only as good as the system, and for the moment the NSG mouse is the best system we currently have to test this.

So until we reach that point, studies into expanding HSCs and progenitors will continue to have merit.

 

Ross La Motte-Mohs • I also agree that improvements can be made to increase the yield of fresh cord blood HSCs without expansion, but there are cases where numbers are low -- some cords are juicier than others and are suboptimal as 1 unit of cord blood.

Also I don't claim that CD34 is the best nor the only marker to use, CD34- mouse bone marrow cells repopulate all hematopoietic lineages. And the lack of that marker is common to MSCs, but to plenty of other cells as well. There are other markers CD133 (PROM1) that serve as HSCs markers. Clinically, CD34 is still the one being used. Is it the right one? Perhaps yes, perhaps no, but it does work in HSCT.

 

Igor Katkov • Philippe,

"WESTERN CONTEMPORARY DIGITIZED MENTALITY prefers 1/0 signal", there are other, "wholistic" approaches that can see the rainbow. Aristotle logics was not the one that had made Homo sapeins different from other hominids. Neither "ing" and "yang" did the work. It had been done long ago, where the first troglodite could grasp the difference between "the single" and "the whole" (NOT PLURAL, elephants can count to 20!). The "Avatar" aboriginals is one of the examples, some indigenous cultures over the World as well.

 

Igor Katkov • Alexey,

Not just "fresh" - efficently frozen but not expanded/tampered in vivo!

 

Igor Katkov • Ross,

I bet you loose 60-70% of their original efficiency during processing and banking no matter how "jucy" the samples are.

Alexey Bersenev • Ross,

The point i'm trying to make is that while we don't know (there is no evidence) can we expand HSCs (Lin-/CD34+/CD38-/CD90+/CD45RA-) or not, we and clinicians and companies should use correct term - hematopoietic progenitor expansion, forget about "stem cell" term. After researchers and companies will show in preclinical study that we can actually multiply HSCs (by phenotype that I indicated and function), everyone can widely use term "HSCs expansion". Incorrect terminology confuse lay public (lay researchers, lay clinicians, lay business guys). So it's our time to discuss this.

CD34+ for human progenitors is good marker, predicative and probably directly correlates with engraftment, but for now it's nothing to do with stem cells. This population contain 90% of progenitors and they should be credited for short-term engraftment and prediction of outcome. It's still speculative, but Bernstein trial support that. You increase CD34+ number --> increase engraftment ---> improve neutrophil recovery --> improve clinical outcome, but HSCs number withing CD34+ wend down or stayed at the same level. That's all clinicians need to know now. And we should tell them - credit CD34+ progenitors number for success.

 

Alexey Bersenev • Ross,

single cell transplant sorted based on surface phenotype does not solve problem of heterogeneity. If you go by mouse HSCs as pure as we can get today Lin-/Sca1+/cKit+/CD34-/Flk2-/CD150+/CD48- , only 1 out of 4 of these guys are long-term repopulating and self-renewing. And even if you can get this 1 out of 4, it still will be myeloid- or lymphoid biased and could be in G0 or G1 of cell cycle and so on. The same thing I'd assume going on in human HSCs. So the only reliable tests we have today is functional transplant assays, with some limitations that you mentioned above. But it's good enough to answer the question: "What are you really expanding?" stem cells (and what kind - short- or long-term) or progenitors (and what kind - myeloid? lymphoid?).

I think physician doesn't need to know all of these big scary signatures (like Lin-/CD34+/CD38-/CD90+/CD45RA-), he/she need to know what the heck are you expanding? So far nobody (research labs or companies) rigorously tested it in preclinical protocols, but everybody claim "we are expanding stem cells!" Of course, "stem cell" sounds "hot and sexy" - it can get you a grant or easier commercialization, but it mislead the field, IMHO.

 

Weiwen Deng • If we want to expand HSC in vitro, we need a stem cell niche similar to the niche HSC needs in bone marrow. I believe that hypoxia is the key. I can recall a company selling hypoxia instrument for MSC culture uses 2% oxygen. Does anybody know the true percentage of oxygen in the bone marrow of healthy person? We know that the air we breathe in is 21%.

 

Ross La Motte-Mohs • @Alexy

We are both in agreement Alexey. Technologies expand mutilple or unique populations in vitro and in vivo nature (age and niche) probably skew the outcomes of both progenitors and HSCs. I've always detested the term "stem cell" or "true" stem cell as others state. The differentiation potential of any progenitor cell whether you call it stem, short-term, long-term etc... has to be taken into context by the functional outcomes and of course gene regulation (i.e.: CD34 and MHC-II). Clearly, something that you and I are well aware of and agree upon. IMHO, Stem cells and progenitor cells can best be referred to as a continuum where we are still filling in the gaps and blanks. We know something about molecular and cellular phenotypes of these cells (progenitor and stem) according to source, methodology of purification, state of development, what progeny they can generate in vitro and in vivo. We also are gaining insight into how supportive stromal elements are in maintaining and promoting their differentiation (ie the niche). What is exciting personally is the association between stem/progenitor cells and niches (bone marrow, thymus, tissue-specific), compounded by state of development, age and niche degeneration. There are still reasons to pursue expansion technologies: whether they have a clinical reason (which is still based on CD34 numbers), grant funding, papers, patents, or fit into a novel business model. And as you elegantly mention there are more questions to answer as to what cells you are expanding and what cells are engrafting. Without a doubt, it misleads people in the field. Definition of the misleading term "stem cell" is slide number 1 in my presentation.

I will still wait on Bernstein's trial (great paper) to see the outcome of T and B cells. He still needs more time to see these numbers recover.

@Igor

I don't doubt that processing and manipulations changes the "nature" of the cells present in CB or BM, nor that you lose plent of cells in the processing. Less manipulation is certainly favored in clinical trials. I'm sure you are continuing to improve on CB or BM processing (debulking, freezing, thawing, etc) and there is ample evidence that CB-HST can be done effectively even 15-20 years later. So it works clinically. Nevertheless, expansion technologies are still being pursued and probably will be in the near future because the problem still is how to treat one adult patient with one unit of suitably matched cord? Yes, yes you give two suitable cords and one eventually wins out. This may eventually become the method of choice but until then, both two cords transplants and expansion technologies will be explored. Most likely both are in the R&D divisions of cord blood banks and in clinical trials within transplant centers. There is no reason why both can't be done in tandem.

@ Philippe

Absolutely right regarding CD34 expression, it's not black and white. It's just a convenient marker that clinicians and researchers use. CD34 use always has to be taken into context, and I'm glad to see you made that point.

 

Ross La Motte-Mohs • Weiwen, Sanyo sells hypoxic incubators and you can set the oxygen temperature to what you want. There are studies out there that give the ppO2 relating to tissue and this depends on anatomical location to capillary beds. HSCs can be grown under hypoxic conditions of 1%, but best to read up and figure out what will work for your system.

 

Jyoti Kode • Has anyone mentioned success story of Delaney et al, in Nature Medicine,Jan 2010 on this forum. They have succeeded, may be others too should not loose hope to crack the jackpot!

 

Dr.M. Chandrashekhar MD • These discussions have touched all aspects of Stem Cell expansion & Iam impressed with the quality & depth of discussions.

 

Igor Katkov • Dr. M.

all aspects of HUMAN HEMATOPOIETIC stem cell expansion. It is not too late to edit ;-))

 

Bernd Giebel • Hi everybody,

good discussion about CD34 as a marker. CD133 is a much better human HSC surrogat than CD34. According to Bathia CD34- SCID repopulating cells are positive for CD133.

We observed that after two or three days in culture CB derived CD34+CD133+ cells split up into two different CD34+ populations, one maintaining CD133 and one with decreased CD133 levels. Of note, cells with LTC-IC potential are strongly enriched in the CD34+CD133+ population and almost completely depleted within the CD34+CD133low fraction.

Bernd

 

Alexey Bersenev • Ross,

Well said. I think most people who is discussing here in agreement.

I think you can not just simply compare 2x unit CB with expanded HSCs even these techniques serve the same purpose. Simply because (1) 2x unit CB almost got to the point of wide clinical use or phase III, but HSCs expansion still not more than experiment, and so far (after 20 years) - unsuccessful experiment; we don't have expansion technique on our hands to compare. (2) There are many other approaches, very different from 2x unit CB, serving the same clinical purpose, I'll list them below.

I'd continue from other way around:

Let's say I'm an entrepreneur or BMT physician and want to pursue a techniques, allowed improved HSCs engraftment and better clinical outcome in BMT clinic. So I have a list of possible techniques which I can pursue. I'm listing also a phase where at this technique now and very rough time projection to complete phase III.

1. 2x unit CB -- phase III- wide clinical use - 1-3y

2. increase yield of HSCs by processing and freezing-thawing --preclinical-phase I - 4-8y

3. split dosages CB transplant - preclinical-phase I - 4-8y

4. pharmacological manipulation of HSCs/progenitor homing and migration - experiment - preclinical 5-8y

5. pharmacological manipulation of BM niche - preclinical-phase I - 4-8y

6. intra-bone BMT - phase I-II - 2-5y

7. co-transplantation with cells increasing engraftment - phase I - 4-8y

8. expansion of hematopoietic progenitors - phase I - 3-6y

9. expansion HSCs? - experiment - 10-20y

So far my list including 9, all of them more or less serve the same clinical purpose. So what option should I pick to start? I'd probably go for first 5. Faster, clear business model, safer.

Why I gave #9 10-20 years to go? Because If I'd be in FDA, I'd ask to long-term outcome (5-10 years) to make sure that it safe in term of carcinogenesis. Because you want to activate self-renewal, but scientists can not uncouple self-renewal and carcinogenesis today.

Ok, let's imagine #2 and #9 completed all of stages successfully in 10 years. Let's imagine they have the same cost and same efficacy. If i'll get sick or my relatives and I have equally options #2 and 9 to choose from, which one you will go? I'll choose #2.

So, probably people want to pursue all 9 today. It's make sense, because at the end maybe 2 or 3 going to work and maybe one will be less affordable for many patients than another. I'm not against it. Go ahead. But I'd not go for 9.

 

Dr.M. Chandrashekhar MD • Point well taken, Igor.

 

Alexey Bersenev • @Jyoti Kode

Delaney - Bernsten's paper in Nat Med was widely discussed here.

Also i posted about it on my blog - http://bit.ly/d7MrWgIt was one of the turning points for me to realize that we probably don't need HSCs expansion.

Follow-up and this discussion linked to -

http://bit.ly/d9HUS9They did not achieve HSCs expansion. They were able to achieve only myeloid and multipotent progenitors expansion, which was good enough for better clinical outcome.

This study actually put necessity of clinical HSCs expansion under the question.

 

Philippe Guardiola • The fashion victim "CD34" is obturing some kind of view of HSC markers used in clinics, like PROM1 and maybe others. Clinicians just think about this single marker which is probably not the best way to evolve on all these concepts...

Dont only think about fastening myeloid recovery with CBT, we also need T cells for infections and GvL effect...

 

Robert Oostendorp • Hi @Bernd:

With regard to tracking true human HSC in culture, check out the latest Peter Zandstra on this: http://www.ncbi.nlm.nih.gov/pubmed/19897585 .

Those of us who work with mouse cells have a natural scepticism for CD34 as a stem cell marker.

 

Weiwen Deng • To my knowledge, MSCs are adherent cells and HSCs are suspension cells. Are HSCs or CD34+ cells really suspsnesion cells? Please share your opinion.Thanks.

 

Ross La Motte-Mohs • In vitro studies support this notion: MSCs are more adherent and HSCs less so, but bear in mind that HSCs express adhession molecules; i.e. CD34 is an adhesion factor, and have remarkable plasticity for localizing to niches and emigrating out of the bone marrow and immigrating (homing) to tissues. It's probably more accurate to state that HSCs/progenitor cells are easier to mobilize. That being said, it wouldn't suprise me that with the right signals, MSCs can be mobilized and modify their adherent properties. The do function after all in diverse environments and yield several lineages.

 

Steve Wolpe • In terms of the MSC/HSC dicotomy, to date, no one has been able to convert MSC to HSC. They may have a common progenitor, but they appear to be different stem cells. With regard to CD34, people have moved beyond it to look at markers like ALDH, SLAM markers (CD150), etc.

 

Philippe Guardiola • Only very few HSC are circulating in the peripheral blood unless there is a blood disorder such as myelofibrosis in which you have an increased percentage of circulating CD34+ cells in the blood. But HSC are supposed to move from the osteoblastic niche to the endovascular niche...at least. We are a dynamic process !

Moving "away" from CD34 for clinicians , when talking about HSC, is not that easy !

I Guess MSC and HSC have different methylomes...so that they cannot go from one to the other ?

 

Weiwen Deng • When I culture Ficoll-separated mononuclear cells from human bone marrow, I found that I have to wait for 1-2 days to see adherent MSCs in culture flask. This suggest that in MSCs do not express adhesion factor human bone marrow. MSCs start to express cell surface adhesion factor during this 1-2 day culture period. If MSCs already express adhesion factor in bone marrow, the cells should adhere to culture flask within several hours after cell plating. Therefore, we should say that both HSC and MSC are suspension cells in bone marrow. Am I right? Thanks.

 

M. Rao Mahendra • Has anyone looked at the recent Mesoblast news item? It suggests that co culture with MSC leads to an increase in the number of engraftable HSC.

If it works then it is a game changer in my mind

 

M. Rao Mahendra • General comment:

The cord blood banking field appears to be big business but private cord banking has a big stigma to it as it is unclear how useful banking is. Hard to get MSC- may get angioblasts and usually don't have enough HSC and pooling has its problems particularly for private banks.

Making iPSC from cord blood or expanding the number of HSC in cord blood could change the perception of that industry don't you think?

I am not affiliated with this industry so have no particular axe to grind

 

Philippe Guardiola • Regarding allogeneic SCT, I think the big thing might not be HSC but getting more T cells from this type of graft to limit infections and improve disease control through GvL effect. In addition this could also have some consequences on engraftment rates.

 

Alexey Bersenev • Rao,

which press-release of Mesoblast are you talking about?

If you look at: http://mesoblast.com/news_mediareleases.phpthere are 2-3 news about CB from HSCs expanded on MSC stroma - trial in MD Anderson.

I've read them, one excerpt:

"Professor Itescu said the proprietary MPCs expanded haematopoietic stem cells in umbilical cord blood by approximately 40-fold. In patients receiving MPC-expanded cord blood, the median time to neutrophil recovery was 16 days and to platelet recovery 38 days, compared with approximately 30 days and over 90 days, respectively, in published reports of patients transplanted with an unexpanded cord. To date, only two patients have developed Grade III/IV graft-versus-host disease, compared with approximately 40% in published reports of patients transplanted with unexpanded cord blood."

http://mesoblast.com/mediareleases/130.pdf

Based on all press-releases we can not conclude anything about HSCs. Same with trial of Bernstein in Fred Hutch, they got improved neutrophil recovery and platelet recovery. It could be explained by progenitors expanded in CB sample. To prove HSCs expansion in human, they need to show that donor's cells persist in BM for 1-2-3 and more years and blood multilineage chimerism coming from expanded sample much higher compare to fresh un-expanded. There is no data on this.

I think, all approaches (8) that I indicated above, directed to increase engraftment and outcome will change a game completely for private banks. If you can double total MNC or CD34+ recovery from 1 cord simply by improvement of cell processing procedure, why do you still need expansion and 2x unit CB?

 

Philippe Guardiola • Alexey,

30 days is not anymore the median time to achieve neutrophil recovery with unexpanded CBT unless you select a "bad CB unit" which is ot what you are supposed to do as a transplanter !

In Barker s paper with RIC it was at a median of less than 10 days, maximum 28 days. Blood. 2003 Sep 1;102(5):1915-9. Epub 2003 May 8.

And it was 23 days for 2 CBT with myeloablative conditioning Blood. 2005 Feb 1;105(3):1343-7. Epub 2004 Oct 5.

Which also suggests that the type of conditioning regimen may be also affecting speed of neutrophil recovery as well as the use of systematic GCSF in some cases.

If we really want to know what is true we will defiintively need a phase III randomized study comparing these different options... Be careful about small studies claiming that they are the best option.

18 patients, let say highly selected ones, as usual, 2 with grade III-IV AGvHD...nothing special here ! Just wait for 50 to 100 patients more and you will get the same results as with other strategies... roughtly 30 to 40% alive and cured !!!

 

Steve Wolpe • Expanding progenitors alone will lead to late engraftment failure when the progenitors burn out and aren't replaced. A recent paper in Blood ( http://www.ncbi.nlm.nih.gov/pubmed/20382848 ) shows that the contribution of short-term progenitors is restricted to the first 5 months. It therefore doesn't take 1-2-3 years to demonstrate HSC expansion. However, the need for expansion of LT HSC depends on the clinical conditioning regimen used. If it's reduced intensity conditioning (RIC) then probably enough endogenous HSC are spared that expansion of progenitors is sufficient.

 

Alexey Bersenev • Steve,

I saw this article, very interesting. Short-term repopulating cells is between true stem cells and multipotent progenitors. It's the first study indicating that function of any (expanded or non-expanded) donor HSCs should be assessed after 5 months.

We know the time point for mice: progenitors - 0-2-3 months, short-term HSCs 3-5 months, long-term HSCs - 4-6 and > months after BMT. How to translate this time points for assessment of HSCs function in human is not clear. So this paper is the first evidence.

We need a lot of progenitors going only first 3 months, than we need short-term HSCs and then finally long-term HSCs. if you can keep your patient happy and alive on progenitors first few months, you can improve outcome, we need HSCs to work after that.

 

Jim Hardy • Alexey, I forgot about this article I saw in January: "First successful use of expanded umbilical-cord blood units to treat leukemia" http://www.lifesciencesworld.com/news/view/130611 

Bernd Giebel • Dear Jim,

it is a great paper, but do they show HSC expansion?

Best

Bernd

 

Alexey Bersenev • Jim and Bernd,

Great paper, agree, clarified a lot of things. HSCs expansion was not achieved by the authors. They showed that we really don't need HSCs expansion for clinical benefit.

We have talked about this paper in this discussion. If you will get time to go through all 100 comments or maybe just through bottom 50.

Also I analyzed this paper here -

http://bit.ly/d7MrWg 

Igor Katkov • It was actually 99 comments before. No specific comments from my side this time, I agree in general with Alexey, and I emphasized the importance of processing and efficinet cryopreservation as the "missing link" before. Just wanted to be the hundredth one ;-)))

 

Igor Katkov • 1. on SCs: has anybody heard work of Marius Ratajczak on VSEL?

INTERE$$$TING!

This one for example:

http://www.ncbi.nlm.nih.gov/pubmed/20629987

Then click on his name and VSEL.

 

Steve Wolpe • Two issues about VSEL's. One is that I've never seen another field of research where the ratios of reviews to primary papers is at least 3:1 the way it is with VSEL's. Second, the only papers I've ever seen on these cells come from Ratajczak or labs closely associated with him. The work is very interesting but it sure would be nice to see it repeated by another reputable lab.

 

Weiwen Deng • I believe that those tiny VSEL-SC are in fact progenitors of both HSC and MSC. We need to figure out a way to differentiate ex vivo expanded VSEL-SC to HSC. Then our discussed issue is gone.

 

Ross La Motte-Mohs • Steve hit the nail on the head. I would add that a third issue that is missing from VSELs is clear functional data (which in the number of years they have devoted to writing reviews should have materialized). It's very nice that VSELs can be isolated from bone marrow, cord blood etc, but one wonder why in this time frame of highly repetitive publications on the purification of VSELs, they have not been shown to at least differentiate into another cell type? Something which can be shown on the single cell level with HSCs.

The abstract is ambitious, but the theraeputical implications for these cells are highly circumspect and weak to claim therapeutic efficacy. This paper's abstract promises much but delivers little. I am intrigued about the notion of a stem cell progenitor upstream of HSCs and angioblasts, but until I see differentiation data from independent labs, I remain unconvinced that VSELs are these cells. My personal opinion is that VSELs are too small to be a true stem cell and the data that has been collected and presented thus far does not rule out apoptotic ghosts/shells. Of course, I could be entirely wrong and this would represent a paradigm shift that stem cells can be smaller than a red blood cell, a cell that has expelled its nucleus and DNA content, but still expresses cell surface markers. There are a number of experiments that Ratajczak could do to convince others of their differentiation potential or functional capacities. The big question is why isn't this being done?

 

Alexey Bersenev • I agree with Steve and Ross.

Firstly - reproducibility in other independent labs. Second - publications bias. Third - lack of functional data - they have to simply apply all high profile assays that we have for HSCs to prove VSEL's stemness. I've heard Marius Ratajczak talk at ISCT and I was not very convinced, because of lack of functional assays

You can also look at my bookmarks collection about VSEL:

http://www.connotea.org/user/nanog/tag/VSEL 

Igor Katkov • Weiwen, they are "EMBRYONIC stem like", not "progenitors", because they express Oct4 and other PLURIPOTENT markers in CONCERT, that is why "E" in VESL comes from.

Steve, Ross and Alexander: Marius(h) is comingt to us on Friday and will be grilled by the audience of die-hard ESC people. I will report what happens and what his arguments are.

Why OTHER labs haven't repeated it yet? Two reasons 1) It is quite new; 2) Huge bias toward Eastern Europeans and Russians; had it been published by a Westerner or Japanese, who knows. Sometimes they miss Sputnik, or stem cells, or TV, or helicopters, or Stealth technology, etc, that way, then re-discover and claim as it is from the Western "pioneers". Sometime they just invite the inventor, give him a bunch of money, and 5 years later he becomes"a great American inventor Igor Sikorsky" or Nobel Prize Winner Prof. Dulbecco. That is what probably U of KY did.

My point is that Marius has to fight hard to get the same attention than a Western scientist would have to get it repeated somewhere else here. So, we'll see whether this time it is a nugget of like just as Noami Kimball said yesetrday "some dirty pebbles" VERY SOON, patience, pls.

 

Steve Wolpe • I doubt that's the case. First, Ratajczak is head of a stem cell institute in Louisville, Kentucky, not some village in Outer Mongolia. Second, look how quickly the world jumped on Yamanaka's discovery of iPS cells. Ratajczak's stuff has been out for about 5 years now and yet no one has reproduced the findings. Doesn't mean he's wrong but it is rather curious.

 

Steve Wolpe • By the way, he gave a talk at the ISCT meeting. I asked him if, when his VSEL's made hemoglobin, if it was fetal or adult. You'd think if they were embryonic-like that they would give rise to cells making fetal hemoglobin. He had no idea.

 

Alexey Bersenev • Yes, VSEL cells being here for a while, longer than iPS. I'd guess maybe about 5-7 years. iPS spread like crazy because is reproducibility!!! Seem like it's not the case for VSEL. Pluripotent markers doesn't make them ES-like. If they claim that VSEL very similar to ES they should demonstrate teratoma-like structures formation, embryo chimerisation in all 3 germ layers and so on.

If i'd be Ratajczak I'd put a protocol for VSEL isolation/characterization in open access and call everyone to try. Or better make a video-protocol and submit to JoVE for free access.

They have couple of protocols actually, I don't know why other labs are not eager to try.

http://mrw.interscience.wiley.com/emrw/9780471142959/cp/cpcy/article/cy0929/current/abstractthis one actually for free -

http://versita.metapress.com/content/q1x283117674317n/Btw there is a company which commercializing VSEL, maybe we can ask them about potency assay of cell product?

I hope Igor will get something interesting for us after Ratajczak talk.

 

Weiwen Deng • I noticed those tiny stem cells in my culture flask 8 years ago when I was a graduate student in Tulane University. I believe that I can figure out a way to culture the cells. But currently I have no funding to do so. What a pity!

 

Jian-Xin Gao • Based on our experience, I would say VSEL cells do exist and are the progenitors of tissue stem cells.

 

Steve Wolpe • Can you tell us about that experience and the supporting data you have?

 

Igor Katkov • I invite everybody to move THIS discussion on VESL to a seprate topic. First, it has quite distant relation to the topic of this discussion oh expanding HMS inititated by Alexey so some here even confused VESL with HSC!

Secondly, it becomes rea-ally annoying clicking those "Show me more comments" 6-8 times to get the last comments, this LinkedIn system sucks because it should/ve shown comments in reverse chronolgy, last comments shown FIRST!

So, Steve, Ross, Alexey, Weiwen, Jian-Xing, please, move this topic a separate discussion I've just started, I should've started it separately at first place, it is still not too late.

 

Alexey Bersenev • Seem like nice article from Cooke -

http://stke.sciencemag.org/cgi/content/abstract/3/134/pe26Would somebody like to evaluate with me?

 

Igor Katkov • I would:

1. Isn't it from Christof Niehrs and Michael Boutros.

2. What does this have to do with HSCs?

Wrong link?

 

Alexey Bersenev • I'm sorry for wrong link.

The correct link -

http://www.sciencemag.org/cgi/content/abstract/science.1191536

Michael Cook study

 

Igor Katkov • Thanks.

Smth weird is going on with the mesages, I wanted to respond HERE ONLY, it ended up in both here and on VSEL site.