VSEL in translation

by Alexey Bersenev on April 15, 2014 · 0 comments

in cell product, other adult stem cells

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Despite the controversy, surrounding VSEL cell biology and methodology, we are keep hearing news about its clinical translation. US-based company – NeoStem, which commercializes VSEL (VSEL is a trade mark of NeoStem), recently announced collaborations with Harvard/ MGH and NIH for VSEL translational studies. Company pumped > $4M into VSEL programs. About 70% of VSEL funding is dedicated to bone regeneration. Last year, NeoStem shared some exciting plans about use of VSEL in bone regeneration:

Phase 2 investigation and first approved NIH clinical study of VSELs™ in humans. Enrollment for this study is expected to begin in 2014.

The required preclinical data, cell manufacturing processes and clinical protocols necessary for submission of an IND to the FDA are in the final stages of preparation. The Company anticipates IND submission in late 2013 or early 2014.

Half of year before this PR, CEO of NeoStem – Robin Smith said:

… we hope to bring into human testing for boney defects in 2013

Well, it did not happen in 2013. Will it happen in 2014? It’s not clear to me, how are they going to start Phase 2 trial without conducting Phase 1 safety study? Did they make a mistake in press release? By this time, they should have approved IND already, but I feel like the process is slowed down. In this post I’m going to analyze “clinical manufacturing readiness of VSEL cells” and highlight possible reasons for delay. This analysis is based on publicly available, published studies by Taichman’s group.

Controversy and reproducibility issues
In order to translate product to the clinic, research protocol should be: (1) highly reproducible, (2) robust, (3) scalable, (4) clinically grade. NeoStem bought VSEL-related IP few years ago from University of Louisville, made “VSEL trade mark” and offer Scientific Advisory Board position to Mariusz Ratajczak. Taking in account such involvement, it’s unclear to me why NeoStem did not make any public comment about ongoing VSEL controversy. However, last year, Robin Smith commented on VSEL controversy in interview:

We acknowledge that the final chapter describing the full potential of VSELs is yet to be written, and that controversy exists at this time regarding how that final verse will read. However, we are proud of our efforts, and those of our collaborators, to get to the truth about these cells, and to apply rigorous scientific method to write the script.

We don’t know if NeoStem ever attempted to reproduce original Ratajczak’s protocol for human VSELs. All published data so far indicate that NeoStem significantly modified original human VSEL protocol and even created their own. So, reproducibility of human VSEL cell isolation is inconclusive as of now. Smith commented:

NeoStem and our collaborators have shown that VSELs are present in the bone marrow and other organs of humans, and can be mobilized from the bone marrow into the peripheral blood and collected by apheresis. It has been established that these human VSELs are comparable to those described by Dr. Ratajczak.

But how much NeoStem’s VSELs are comparable to Ratajczak’s VSELs? And how much they are comparable to other stem cell populations in human bone marrow? In the last few articles Ratajczak emphasized:

… we have always emphasized that, in contrast to their murine counterparts, much work still needs to be done to better characterize human VSELs.

Recently, Ratajczak’s group published “the proper criteria” for identification of VSEL, citing cord blood as a source of human VSELs, not bone marrow. However, NeoStem is commercializing exclusively autologous bone marrow VSELs.

So, (i) if much more work should be done in order to characterize human VSEL (especially from bone marrow) and (ii) if reproducibility of human VSEL protocol is unknown, are they ready for prime time?

Manufacturing steps and cost estimation
Based on last studies, published by Taichman’s group, we can take a look at some piece of “clinical protocol” for VSEL manufacturing. Please note, that VSEL “manufacturing” was done by NeoStem staff in their lab, therefore it’s a “clinical prototype”. Human bone marrow VSELs isolation procedure includes:

  1. mobilization protocol (hrG-CSF, pre-apheresis cost ~$1.5-3k)
  2. apheresis (~$3k)
  3. elutriation (~$1k)
  4. positive selection by MACS for CD34+/CD133+ cells (in case of CliniMACS cost ~$2-5k)
  5. FACS sorting
  6. cryopreservation

So, NeoStem VSEL protocol can end up with manufacturing cost ~$10-15k at least. If we look at main competitor of VSEL in clinical bone repair – mesenchymal stromal cells (MSC), we can estimate manufacturing cost as few hundred $ for allo- and maybe few thousand $ for auto- expanded MSCs. Interestingly, Taichman wrote:

… there are potential limitations to the use of autologous MSCs in bone repair in humans because most preparatory protocols require the extensive expansion of MSC populations in vitro using animal-derived or recombinant growth factors as well as modulators of transcription and cell survival.

My estimation does not support this claim.

Questions to Taichman’s study
The most important translational step for human VSEL was made with recently published study by Taichman’s group. Media called this report as “compelling evidence” for multipotent VSELs in human. I have some questions and concerns about interpretation of Taichman’s study.
VSEL sorting (FACS):

  • There is no discrimination of aggregates (singlets versus doublets) on presented flow plots – it is extremely important for identification of tiny cell populations.
  • Too many dead cells – they showed a plot with 7AAD staining with 40% dead cells before FACS sorting – isn’t it too much?
  • Did MACS really work? They showed a plot with only 24% of CD34+/CD133+ (gated as alive Lin-) after positive selection for these 2 markers – it is way too low.
  • What was in Lin antibody cocktail? They didn’t indicate it clearly, but mentioned that cells were “gated for Lin- Gycophorin A (CD235a)- CD41-“. Were CD235 and CD41 included in Lin cocktail or used separately? What else was in Lin cocktail? It is very important!
  • Different groups employed different gating strategies for human VSEL – so, reproducibility is inconclusive.

Multipotency interpretation:
Multipotency of human VSEL was concluded, based on expression of genes, typical for 3 germ layers (osteocalcin, nestin, insulin), after addition of different inducers of differentiation. This is not a proper test for multipotency. Artifacts should be ruled out in vitro and function of VSEL-derived mature cells should be assessed. It is unclear whether those markers will be expressed without co-culture VSELs with C2C12 line. It is unclear to me why, on the one hand, VSEL made bone tissue after subcutaneous implantation, if there was no indication for their induction in vitro, but on the other hand, VSEL expressed markers of multiple tissue in bone defect site. Shouldn’t it be vice versa? Don’t we need only bone in bone defect site?
Controls:

  • Analysis of normal blood for VSEL from unmobilized donors is not presented.
  • For 3 germ layers gene expression controls with VSEL, cultured in basal media (without inducers) should be used. It is known, that some cell populations can spontaneously over-express atypical markers in culture.
  • MSC, usolated from the same donors should be used as control for bone defect experiments in order to show advantage of VSELs.

VSEL cells fresh or cultured?
Unlike any other stem or progenitor cell population, VSEL cells do not proliferate in culture. In order to overcome this problem and test “multipotency” in vitro, Taichman’s group used C2C12 line (mouse myoblasts) to co-culture human VSEL. The most important clinical questions would be:

  1. How much VSELs is enough for therapeutic effect?
  2. Do VSEL need to be induced in vitro before administration?

The first question can be tackled after safety trial, but the second question should be answered before. My understanding is that NeoStem will got for freshly isolated (not cultured) and cryopreserved VSELs.

Scalability
If we do the math, based on flow plots from Taichman’s study, we can estimate about 0.0002% of VSEL cells in positively selected (for CD34+/CD133+), elutriated, mobilizhed apheresis product. Taichman noticed, that they typically isolate ex vivo, I’d conclude that this technology is not scalable for now.

My another big concern is related to quality control (QC) assays. If NeoStem will able to isolate 1M VSEL from 1 patient maximum, how many aliquotes of cells with how many cells can they retain for QC? Is it even doable?

Clinical grade
Because NeoStem claims start of VSEL trial this year, the protocol should be 100% clinically-grade. However, based on Taichman’s data, we cannot conclude clinical readiness. The biggest question is FACS sorting. As of today, the whole VSEL protocol based on FACS sorting. However, there is no clinical-grade FACS sorter, approved by FDA. Modification of existent instruments can, probably, justify requirements for phase 1 trial, but not more. The second, related issue is clinical-grade reagents – antibody for FACS sorting. In published studies, NeoStem used AutoMACS Pro for magnetic positive selection, but it is not clinical device.

Concluding remark:
I think, NeoStem should do more work for human VSEL cells isolation, characterization and tune up clinical manufacturing protocol. It will take some time. I think, at this point, human VSELs are not ready for clinical translation.

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