As many of you know, I’m highly interested in this topic and proposed a thread “Do we really need hematopoietic stem cell expansion for clinical use?”. I’ve got only one comment on the blog, but 117 comments on LinkedIn (International Society for Stem Cell Research group) and about 10 comments in Google Buzz. The discussion was very useful and interesting. I’ve learn a lot. The field is very dynamic and I’d like to summarize very recent trends today.
Discussion “Do we really need hematopoietic stem cell expansion for clinical use?”
Unfortunately discussions on LinkedIn is not public and I can’t give a link, you have to be “linked in” and you have to be a member of a group. Because the discussion was so big and valuable, I decided to archive it and share it by simple copy-paste in Google Docs.
The result of this discussion is an answer to my question: Do we really need it in clinic? The answer is YES, but:
– if we can really make it (didn’t achieve it so far)
– we should realize that hematopoietic cell expansion ex vivo is just one out of about ten other approaches dedicated to increasing stem cell engraftment and improving clinical outcome. Some of them have reached phase II of clinical trials. So, we should develop all of them simultaneously and pick the ones which are going to work the best, easily reproducible, scalable and affordable.
What do we expand ex vivo? Hematopoietic stem cells (HSC) or hematopoietic progenitors (HP) or hematopoietic stem/ progenitor cells (HSPC)?
We have some experimental evidence that we can maintain and ex vivo expand mouse HSC, but they weren’t translated into the clinic. We just got the evidence that we can maintain and expand human HSC using small molecules in preclinical settings. But, as I said before, we don’t have clinical evidence for HSC expansion yet. So I’d like to propose to use more correct term in clinic – hematopoietic progenitor expansion.
I don’t think we can say: Cord blood has a limitation of use in adults because of lack of HSC per sample. We should say: because of lack of progenitors per sample. We don’t know how many HSC are enough for good clinical outcome and maybe their number per cord blood sample is over bone marrow niche capacity. We know how many CD34+ cells is enough. But more than 90% of CD34+ cells in adult bone marrow and 99% in cord blood are progenitors, but not stem cells. So, very good short-term outcome and rapid multilineage chimerism after transplant clearly correlate with progenitor number.
We can claim HSC expansion only after proper assays. I listed some here. Unfortunately, to-date there is no study, which has rigorously tested the expansion of real human HSC by all necessary assays. The closest study that I was able to find was very recent – Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.
I’d point to the most important assays below:
- 1. Evaluate phenotype of HSC (CD34+/CD38- or Lin-/CD34+/CD38-/CD45RA-/CD90+) or HP (CD34+ total and subsets) before and after expansion;
2. Assess of in vivo repopulation ability in xenotransplant models (NOG; NSG) fresh cells versus expanded taken from one sample;
3. Calculate frequency of expanded HSC in vivo – limiting dilution transplantation assay – fresh cells versus expanded taken from one sample;
4. Self-renewal function of long-term expanded HSC in serial xenotransplant models (at least 2-3 rounds) – fresh cells versus expanded taken from one sample.
What population to expand? Positive versus negative selection
Potential candidates: CD34+, CD133+, Lin-, ALDH+, total mononuclear cells (MNC). The most frequently used approach is positive selection of CD34+ cells by MACS and their culture. Disadvantages of this approach:
- 1. We don’t know how antibodies affect function of cells. There is some data, that CD38 antibody can affect engraftment in xeno-transplant.
2. We lose some positive cells in the MACS column.
3. Positive selection cuts off some accessory cells from total MNC, which could promote HSPC expansion.
4. Using CD34 positive selection, we lose all other HSPC from Lin- or total MNC. There are very potent HSC outside of CD34+ population.
It seems like the best candidate for expansion could be neganivly selected Lin- population, which should cover almost all HSPC = CD34+ and CD34-. But Lin- population still can be depleted for the accessory cells in total MNC, facilitating engraftment.
A recent report indicates that the presence of some accessory cells, such as CD3+ and CD14+ could inhibit HSPC expansion. So, another good option – total MNC, after depletion of CD3+ and CD14+ cells.
Cell culture conditions
- 1. Timing is important. Shorter time is better for HSC maintenance, longer time is better for progenitors expansion. Long cell culture period is expensive, labor consuming and may introduce additional risks – tumorigeneity and immunogeneity.
2. Bone marrow niche mimicking by co-culture with stromal or other cells.
3. Hypoxia is the key to maintenance of stemness.
Preliminary conclusions of two ongoing clinical trials
Two ongoing clinical trials (Fred Hutchinson Cancer Center and MD Anderson Cancer Center) that I like a lot and frequently link to, showing the value of progenitor expansion for rapid neutrophil chimerism and patient recovery. Preliminary conclusions from these trials are the following:
- 1. Expanded CB unit doesn’t sustain long-term repopulation and is always overtaken by unmanipulated unit, which dominates the engraftment (Irwing Bernstein, 2010, Elizabeth Shpall, 2008-2010)
2. Total CD34+ number is expanded and correlates with rapid engraftment and improved neutrophil and platelet recovery
3. Stem cell expansion was not achieved
4. No engraftment failure observed and there was clear clinical benefit
5. We should continue to pursue expansion approaches, but focus on hematopoietic progenitors
Strategies for progenitor expansion
I’d like to promote the concept of hematopoietic progenitor versus HSC expansion. Currently we don’t have specific clinical protocols dedicated to progenitor cell expansion. We’re getting massive progenitor expansion within CD34+ population as not expected or even “side effect” of HSC expansion protocols. The specific approaches could be the following:
1. Expand particular progenitors by applying specific cytokine combination in total MNC or CD34+ selected or Lin- cells.
2. Sort or enrich specific progenitor subsets by cell separation techniques and then expand them.
3. Differentiational mobilization of progenitors from bone marrow with following apheresis and expansion.
Combination of progenitor expansion with techniques increasing HSC engratment and homing
In the near future we can combine expansion of progenitors with pre-transplant treatment of cord blood MNC with agents increasing HSC engraftment. Potential candidates for such treatment could be:
Clinical trials using these agents are already under way.
The possible scenarios of these combination could be the following:
1. One CB unit frozen in 2 equal bags: the first to undergo expansion protocol, the second to thaw later and to be treated a few hours before transplant by engraftment increasing agents.
2. Two units of CB: one expanded, one treated.
3. Transplant of fresh or/and expanded CB unit following injection of engraftment increasing agent.
I think the future of hematopoietic progenitor expansion is bright and promising. The future of HSC expansion looks too blurry to me, I remain skeptical. I believe that there is nothing better that fresh unmanipulated HSC. Any exposure of HSC, taken out of bone marrow niche context, to cytokines or ambient air lead to rapid loss of stemness, which could impair their function and introduce additional risks. I have a gut feeling that the number of true HSC in one good sample of cord blood is enough for rescue of hematopoiesis in an adult patient. We don’t need a lot of them. We have to focus on other techniques for increasing HSC engraftment with combination of progenitor expansion.