I don’t know why, but people still bet on who will inject human embryonic stem cell (ESC) derived cells for the first time ever into a patient - Geron or Advanced Cell Technology (ACT)? Geron’s phase I clinical trial is still on hold and while we are expecting an answer from the FDA , none of the patients were officially treated yet. ACT filed an IND application to the FDA for first human clinical trial using ESC to treat eye disease and hope to start it this year. So, everybody is taking a deep breath before these 2 clinical trials will start officially in US.

Excerpt from Knoepfler Lab Stem Cell Blog:

Still no news from Geron on that clinical hold by the FDA that is now nearly a year long. I’m told (no big surprise) that ACT investors are hoping the Geron hold is very long and that ACT will become the first biotech with an FDA-approved hESC-based clinical trial. I still think Geron will be first but haven’t heard anything in a while.

Meanwhile, countries such as India do not have an FDA and such trials are regulated differently. You may be surprised to know, but more than 4 years ago the first patient ever was injected by ESC-derived cells in the Institute of Transplantation Sciences, India. More than that, results of this experiment were published in a well respected peer-reviewed journal Transplantation Proceedings. We were so excited about this remarkable event that we started to discuss online:

This paper describes a case report of tolerance induction in a male kidney transplant recipient, in which donor bone marrow (from his sister) was “superpotentiated” by the co-administration of embryonic stem cells generated from the sister that were administered with the bone marrow.

The recipient appeared healthy at 100 days post procedure and did not require immune suppression, which is a fundamental advancement in the field of transplant immunology.

I’d like to bring up a methodological part, described in the paper:

We planned to subject her to long ovarian stimulation simultaneous with our regular tolerance induction protocol. The retrieved oocyte would be denuded and enucleated using an aspiration technique. We planned to use a single cumulus cell from the same donor to inject into the enucleated oocyte (somatic cell nuclear transfer [SCNT]-oocyte). The SCNT-oocyte would be developed into a blastocyst. The inner cell mass hatched from this blastocyst would be cocultured with her own unmodified BM for directed differentiation into HSCs. We planned to analyse these cells for their counts, viability, CD34+/45+ counts, as well as to detect molecular markers for pluripotency and karyotyping. We planned to infuse them into the periphery of recipient after confirming their counts and viability. Renal transplantation was to be performed 1 week after the cell infusion…

The Institutional Review Board approved the study protocol and informed consent forms.

Dr. Trivedi, who was brave enough to perform this procedure for the first time in clinic, appeared to be a very nice guy and kindly replied to our request about follow-up of this patient and their future plans:

In response to our coverage of this event, the senior author of the paper, Dr. H.L. Trivedi has requested us to post the following:

” Thank you for your interest in our work. I would like to draw the attention of your readership that this patient will be finishing 1 year without any rejection/ teratoma. He is doing well. We have also implemented the same protocol in 35 more patients successfully. H.L. Trivedi “

Now, 4 years after, I was curious enough to ask Dr. Triverdi about follow-up of this trial via email. Especially I was curious about safety of procedure and I got the following answer:

“We have now abandoned hESC since they were not so potent as thought to be. In fact now we use human adipose tissue derived mesenchymal stem cells which we have patented and these show wonderful results. We have been able to treat renal Tx patients with these cells and our patients do well with minimum/ no immunosuppression.

None of the patients has developed any tumor masses etc, because we differentiated cells into hematopoietic cell lines before transplanting them in to recipients. Many thanks for interest in our work. H.L. Trivedi”

Well, still a lot of questions remain, because they didn’t publish follow-up results (even if it was not clinically beneficial) after this clinical case. But I think this first paper in Transplantation Proceedings is very important, because its claim is a first (is that what you meant?). I was really thrilled by the fact that the first clinical use of ESC-derived cells was done with tolerance induction in organ transplant purpose and that it was with hematopoietic derivatives. The most important message is that, despite the fact that undifferentiated ESC (hematopoietic cells were not purified from cultured ESC) were injected, the therapy appeared to be safe long-term. For some reasons I feel happy for my Indian colleagues, maybe because I spent some years of my life in transplantation medicine.

this is re-blog from Stem Cell Assays

Recent editorial note by Darwin Prockop in Molecular Therapy journal highlights the problem of potential development of malignancy as a complication of cell therapy. We are coming back to this topic again, because new interesting data has come up.

As a background for this caution I’d like to remind you that in 2005 Rubio for the first time reported spontaneous human mesenchymal stem cell (MSC) transformation after standard 6-8 weeks expansion ex vivo. Since that, nearly 10 more reports were published confirming spontaneous adult stem cell transformation in long-term cell culture in mouse and human. It was the first big alarm for cell therapies based on transplantation of ex vivo expanded adult stem cells. Intriguingly, later we got a few reports showing that expanded MSC can not overcome “senescence crisis”, are stable in long-term culture and do not undergo spontaneous transformation.

So, data from different laboratories appear to be very different and conflicting. But the importance of this question is tremendous and worth more than the 100 clinical trials right now assessing the therapeutic efficacy of MSC in different conditions. This problem also highlights the weakness of assays and necessity of developing new “tumorigeneic safety criteria” for therapeutic cell products.

As a follow-up of this intriguing story, two international teams recently reported (here and here) that MSC cultures, which were previously described as spontaneously transformed, were actually cross-contaminated by malignant cells from human cancer cell lines. So, human MSC spontaneous transformation is nothing more that cell culture artifact, as noted by authors.

…we did DNA fingerprinting and/or short tandem repeat (STR) analysis comparing the normal MSC with their transformed counterparts. The analysis shows that the transformed mesenchymal stem cells (TMC) in one laboratory were cross-contaminated with human fibrosarcoma or osteosarcoma cell lines, whereas in the other laboratory cross-contamination was due to two glioma cell lines.

Now, after all these new findings, Dr. Prockop is emphasizing that we don’t have good assays to test contamination of cell product (graft) by malignant cells. More than that, we even don’t know what the probability of these events in cell culture is and how many malignant cells is enough to cause clinical disease onset after cell transplant.

Unfortunately, none of our current technologies provides a definitive test for the presence of small numbers of tumorigenic cells in the large doses required for most therapies.

The limits of our tests for tumorigenicity are severe. There are no hard data on the minimum number of tumorigenic cells necessary to produce tumors in patients, but observations with hematopoietic stem cells suggest that the number could be approximately 100 cells.

He did some calculations and came up with a number:

Therefore, a conservative estimate is that we need an assay that will guarantee that a preparation of therapeutic cells contains less than 1 tumorigenic cell per 3.5 millions.

and more on lack of assays:

Our present assays fall far short of this level of sensitivity. Classic karyotyping detects only major rearrangements of chromosomes, is subject to cultural artifacts, and samples only a small aliquot of any cell preparation. Tests for tumorigenicity in mice are meaningful only if positive, because many human tumors will not produce tumors if directly injected into immunodeficient mice.

What is the possible consequence for cell therapy? Well, we need more assays and more safety criteria now.

Therefore, the field of MSC research has rediscovered the risk of cross-contamination of cell cultures posed by malignant cells, a danger that has been known for many decades but one that still plagues the field of cancer research.

Some new assays and actions, which should eliminate these errors in future, were proposed:

First, because human errors may occur in any laboratory despite stringent working procedures, DNA fingerprinting should be compulsory for all experiments involving cell lines. Scientists should verify the cell lines in their possession and use electronic databases of authenticated DNA profiles against which they can compare their results. Second, scientific journals should require that all cell lines used in an article are verified before publication.

So, the new data showed that spontaneous transformation of expanded human MSC is more likely cell culture artifact. But it gave us a new danger - potential contamination by malignant cell lines, which should be very carefully assessed in cell product development and release.

Connotea tag: adult stem cell transformation

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also read: A delicate balance between therapeutic cell expansion and cancer

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”.

Hundreds of articles have proposed pre-clinical and clinical protocols for so-called “hematopoietic stem cells (HSCs) expansion” and some RegenMed startup companies started to commercialize it. I think it’s worthless to continue to do that.

Ok, now I’ll tell you why I think so and how I came to this conclusion.

1. As soon as clinical trials started we realize that unlike preclinical data we actually can not achieve expansion of engraftable long-term HSCs

The most convincing evidence came from results of a recent trial in the Fred Hutchinson Cancer Research Center. We can expand different progenitors, maybe short-term HSC, but not long-term HSC. The authors didn’t observe any “stem cell activity” from expanded cord blood sample in patients a few months after transplant.

…virtually all expanded CD34+/CD38- cells were multipotent and myeloid primed progenitors with very very few true HSC. Those very few expanded HSC, the authors were not able to detect a few months after transplant in 8 out of 10 patients. 2 patients with some signs of expanded long-term HSC engraftment were under observation for 240 and 180 days. But in one of them they were not able to detect signs of multilineage engraftment at the time point of 1 year and second patient didn’t possess engraftment in T-cells.

2. In order to achieve significant clinical improvement we don’t need a lot of HSCs but we need a lot of progenitors

Bone marrow transplant (BMT) patients are struggling with neutropenia, delayed neutrophil recovery and thrombocytopenia in the first month after the procedure. Toxic myeloablative treatment and absence of neutrophils makes them very sensitive to infections and prone to sepsis. So, the first thing that we need is earlier and rapid neutrophil recovery! Long-term engraftable HSCs are not going to make neutrophils in the first month after transplant, but donor’s progenitors will do it and allow the patient to survive. Multipotent or myeloid or granulocytic progenitors - doesn’t matter, but we need a lot of them. Fortunately it’s much easier to expand them than HSCs.

For illustration of the importance of progenitors for hematopoiesis recovery from irradiation and survival I’d like to refer you to the earlier study of Koichi Akashi.

To identify the principal components of the hematopoietic system that are radioprotective, we transplanted lethally irradiated mice with purified progenitors: common myeloid progenitors (CMPs), megakaryocyte/erythrocyte-restricted progenitors (MEPs), or granulocyte/monocyte-restricted progenitors (GMPs). CMPs and MEPs but not GMPs protected mice in a dose-dependent manner, suggesting that erythrocytes, platelets, or both are the critical effectors of radioprotection.

All animals radioprotected for 30 days subsequently survived for at least 6 months post-transplant, and showed only host-derived hematopoiesis after 30 days. These findings suggest that rare hematopoietic stem cells survive myeloablation that can eventually repopulate irradiated hosts if myeloerythroid-restricted progenitors transiently rescue ablated animals through the critical window of bone marrow failure.

3. T-cells alone, without engraftable HSCs can induce graft-versus-tumor effect and eradicate neoplasm

T-cell therapy (donor leukocyte infusion (DLI) or adoptive T-cell or CAR’s) in leukemia clinic achieved significant progress in the last few years and got close to the point when one can ask: “Can it replace HSCs transplant?” If you think about it, why do we need donor’s HSCs at all? If conditioning regimen is not killing all host normal HSCs, if donor’s (or autologous CAR’s) T-cells can efficiently induce graft-versus-leukemia effect and eradicate leukemic stem cells, we don’t need BMT. I believe that in the future, at least for some indications, cellular immunotherapy alone or with a few normal fresh HSCs will be efficient enough to eradicate leukemia.

As a clinical example I’d like to refer you to some observations, which indicate that clinically significant graft-versus-leukemia effect can be achieved without sustained donor engraftment.

4. Methods for HSCs engraftment improvement will be more clinically sufficient than chasing for increasing of HSCs number through expansion

The problem is not as much in low number of HSCs in fresh cord blood sample as in their low rate of homing to bone marrow niche and engraftment. Would you agree that manipulating HSCs by migration, homing and engraftment after transplantation is an easier and more efficient way to improve the clinical outcome compared to HSCs expansion ex vivo? I believe so, because there are a number of smart approaches for increasing engraftment of HSCs in preclinical development and different stages of clinical trials.

Especially, I like approaches dedicated to the improvement of homing of transplanted HSCs and niche manipulations.

5. Cost, scalability and commercialization

Have you ever thought about how much money and human resources you may spend for clinical “HSCs expansion manufacturing”? You should include in counting: 2-4 weeks of large-scale cell culture (medium, supplements, labware) in GMP facility, technicians work and “product release tests”. Long ex vivo manipulations, risk of cell product contamination and safety tests, included in strict “release criteria” will make it hard to go smoothly through FDA (or other regulatory agency) approval. But if you work with approved substances (such as PTH for instance) in order to increase homing or engraftment of fresh HSCs, you will get easier and quicker approval.

Concluding remark:
I just shared some of my thoughts on “necessity of HSCs expansion for clinical use”. I think that we should re-direct our efforts to:

- manipulations of migration and engraftment (niche interaction) of fresh HSCs;
- different progenitor populations expansion;
- cytotoxic T-cell and NK-cell therapy for graft-versus-tumor effect induction.

We probably should stop writing proposals for “HSCs expansion magic”.

I’d be happy to hear any critical comment and to discuss this further. I hope to hear the opinion of bone marrow transplant physicians on this problem, especially.

Dear readers, I’m very very pleased to share with you great news - Hematopoiesis won the 2010 HAL Medical Blog Awards in nomination ” Future Leaders of Biomed”!

I was very surprised to find the confirmation letter from HAL in my emailbox.

About Future Leaders of Biomed – Best Blog Award:

This award recognizes the top blogs covering biology and medical issues that are by graduate students with exceptional promise.

These bloggers exemplify the hard-work, dedication and skill it takes to make a difference in the areas of biology and medicine.

HAL Medical Blog Awards about Hematopoiesis:

This blog provides breathtakingly detailed and informative coverage of blood stem cell topics. The astonishing depth of its writing makes it a winner, and our number one choice overall.

It is real honor for me to be nominated and recognized as the Best Medical Blog 2010 and to be in the same list with other scientific and medical bloggers, which I know and follow. The full list of winners and more about this Award you can find here.

I’d like to thank the sponsor - Apredica for support this initiative. Also I’d like to thank “Health and Life” for organization of this event, everyone who contribute to Hematopoiesis and all my readers. Stay tuned!

I continue to highlight pathological findings from autopsies and biopsies performed on patients underwent cell therapy procedures. I decide to make a new category - “Stem Cell Autopsy”. It doesn’t mean that I’ll highlight only stem cell transplant cases, but I’ll cover all cell therapies in general.

I believe that information from pathological findings will allow us to gain knowledge and avoid possible complications (sometimes fatal) in designing new clinical trials. To get a sense of how it is important, you can read:

Fatal complications of autologous cell therapy;
Clinical neurotransplantation of fetal tissues - analysis of histological findings;
Potential risk of tumor formation from adult stem cell therapy could be underestimated.

Today I’ll highlight 2 cases:

1. Angiomyeloproliferative kidney lesions following autologous stem cell therapy

This news blew up in mass media last week. A woman, who suffered from severe lupus nephritis underwent autologous hematopoietic stem cell transplant (HSCT) into the kidney in a Thailand clinic and eventually developed fatal complications.

There was no improvement in renal function and the patient began hemodialysis 3 months after stem cell therapy. Six months after therapy, the patient presented with left flank pain and hematuria. Ultrasound and magnetic resonance imaging studies showed a 4.0-cm enhancing mass in the left renal pelvis, with smaller lesions in the left kidney, the liver, and right adrenal gland.

The clinical impression was urothelial cell carcinoma with metastatic spread to the right adrenal and liver. The patient underwent a left nephrectomy 11 months after stem cell therapy. The patient continued on hemodialysis over the next year but gradually deteriorated and died of sepsis after infection of the arteriovenous shunt.

The correlation between stem cell treatment and cause of death is unclear, because an autopsy was not performed. All conclusions were based on pathological examination of the removed kidney. So, we can’t claim “death caused by complications of stem cells treatment”.

Thus, the lesions in this patient showed both angioproliferative and myeloproliferative components, with an appearance not typical of any tumor or reactive condition. Given thatthere are multiple lesions in the left kidney and this patient received multiple injections of stem cells into this area, the logical conclusion is that these lesions are stem cell– derived or stem cell–induced. Because the exact number of injections is not known other than “multiple,” the number of lesions in the left kidney may or may not have exceeded the number of injections. We cannot rule out that one or more of the smaller lesions could have spread from the main lesion. Although the lesions in the liver and right adrenal were never biopsied, we postulate that these are the same as those in the left kidney. All injections were given blindly and the liver and right adrenal may have been injected instead of the right kidney, particularly because no lesions were detected in the right kidney.

Importantly, we can’t call these lesions a tumor, but should instead use the term proliferative disease.

Although the histology of these lesions appears to be benign, it is unknown if these are truly neoplastic or localized proliferations of normal stem cells. If they are stem cell -derived or -induced, the proliferative and recurrence potentials are unknown and the classic concepts of benign and malignant may therefore not apply.

My 2 final points:
- It is unclear whether these lesions arose from hematopoietic stem cells or more mature progenitors;
- it is unclear whether lesions were made of transplanted mobilized HSC or brand new cells migrate from bone marrow in site of tissue injury.

2. Neuroinflammation and demyelination in multiple sclerosis after allogeneic hematopoietic stem cell transplantation

Authors examined the histopathological findings of 4 multiple sclerosis (MS) patients who received allogeneic HSC transplantation and died from different reasons.

Four patients with MS who died at a median of 4.5 months (range, 3-9 months) after allo-HSCT for a concomitant hematologic malignant neoplasm; 5 patients without MS who died at a median of 10.0 months (1-29 months) after allo-HSCT; and 5 control subjects without MS who did not undergo allo-HSCT

The present study is, to our knowledge, the first to examine CNS histopathological findings in MS patients after allo-HSCT. Despite the procedure, active demyelination and inflammation persisted, indicating the failure of allo-HSCT to halt the MS disease activity, at least during the follow-up period in these patients.

Although the non-MS brains that underwent allo-HSCT contained diffuse but mild inflammation, no obvious demyelination was identified in the present study.

In conclusion, the demyelinating and inflammatory activities of MS persist after allo-HSCT. The demyelinating activity is presumably due to the persistence of recipient immune cells in the MS brain, whereas the inflammatory activity is more likely the result of GVH reaction after allo-HSCT. The findings of the present small series of MS patients indicate that allo-HSCT fails to stop the demyelination and inflammation of MS.

This is a very well done study. The authors discuss possible mechanisms and compare with other autopsy-based studies, such as after autologous HSCT. Unfortunately, pathological findings after both autologous and allogeneic HSCT confirmed the notion that we have no chance to fix a patient when severe demyelination and neurodegeneration in progressive MS occurs. We should focus on trials in inflammatory stages.