ISSN 1866-8836
Клеточная терапия и трансплантация

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Volume 5, Number 2
06/01/2016 03:09:00 pm
Volume 5, Number 2
Editor-in-Chief
Afanasyev B. V. (St. Petersburg, Russia)
Co-Editors-in-Chief
Wagemaker G.(Rotterdam, Netherlands)
Zander A.R. (Hamburg, Germany)
Deputy Editor
Fehse B. (Hamburg, Germany)
Managing Editor
Chukhlovin A. B.(St. Petersburg, Russia)
Editorial Board
Aleynikova O. V. (Minsk, Belarus)
Borset M. (Trondheim, Norway)
Büchner Th. (Münster, Germany)
Chechetkin A. V. (St. Petersburg, Russia)
Fibbe W. (Leiden, Netherlands)
Galibin O. V. (St. Petersburg, Russia)
Hölzer D. (Frankfurt a.M., Germany)
Klimko N. N. (St. Petersburg, Russia)
Kolb H.-J. (München, Germany)
Kröger N. (Hamburg, Germany)
Kulagin A. D. (St. Petersburg, Russia)
Lange C. (Hamburg, Germany)
Mamaev N. N. (St. Petersburg, Russia)
Mikhailova N. B. (St. Petersburg, Russia)
Moiseev I. S. (St. Petersburg, Russia)
Nagler A. (Tel-Aviv, Israel)
Nemkov A. S. (St. Petersburg, Russia)
Paramonov I. V. (Kirov, Russia)
Roumiantsev A. G. (Moscow, Russia)
Savchenko V. G. (Moscow, Russia)
Smirnov A. V. (St. Petersburg, Russia)
Uss A. L. (Minsk, Belarus)
Zubarovskaya L. S. (St.Petersburg, Russia)
In this Issue
Alexey B. Chukhlovin This issue of CTT begins with a mini-review by Joel Glover who presents a current view on the stem cell seeding and growth in 3D-cultivation systems. The newly developed artificial or natural substrates allow of supporting morphological and functional integrity of specialized cells, including haematopoietic cell populations. Sufficient attention is given to technical issues of 3D bioprinting which is a “hot point” of modern biotechnology. This approach is quite promising, e.g., for in vitro studies of hematopoietic microenvironment, like as for design of artificial organs. Next article (S.Bondarenko et al.) deals with different factors affecting post-transplant survival and relapse rates in patients with acute lymphoblastic leukemia. Of sufficient interest are the single-center data concerning efficiency of donor lymphocyte infusions post allo-HSCT, including a large series of haploidentical transplantations. An analytical review by E. Morozova et al., together with presentation of two clinical cases of primary myeloproliverative disease (MPD) is dedicated to search for optimal treatment regimens for this chronic fatal disorder which was incurable until last years, including Lenalidomide or pegylated interferons. Recently, Ruxolitinib and other JAK2 inhibitors have been proposed for targeted therapy, either alone, or as a supplemental treatment to allogeneic HSCT procedure. A team of heart surgeons (A. Nemkov et al.) has a 12-year experience in cellular therapy of coronary artery disease with local infusions of autologous bone marrow cell (ABMC). They note that the favorable cardiac effect of the treatment fades away 3-4 years later. Repeated ABMC injections cause additional positive actions in term of functional improvement of myocardium, thus presuming possible correction of endothelial dysfunction in cardiac tissues. An article by M. Shaheen et al. reports additional adverse effects of increased pre-transplant serum ferritin upon general survival and complication rates in the patients undergoing HSCT. These data of a single-center study are in accordance with similar works performed worldwide showing, e.g., increased rates of infectious conditions, however, without correlations with graft-versus-host-disease incidence. A study by C. Lange et al. presents a summary of effects produced by the bone marrow-derived mesenchymal cells (MSCs) in irradiated animal model, as seen from increased survival and faster hematopoietic reconstitution. Only small portion of MSCs was revealed in bone marrow. Moreover, MSCs could differentiate into endothelial presurcors, thus presuming a paracrine mechanism of their actions upon hematopoiesis. The article by T. Parkhomenko et al. presents an interesting example of biophysical approach applied to assessment of cell population quality. The authors address an issue of integral energy potential of stored bone marrow cells. A potential- sensitive fluorescent probe was used for evaluation of appropriate time-dependent changes. To implement this technique, some comparisons with conventional tests will be necessary (CD34+ cells, Aldefluor test etc.).

Clinical articles

Impact of initial serum ferritin on early post-HSCT complications: a single-center study

Mostafa Shaheen,1,2 Maria O. Ivanova,1 Ivan S. Moiseev,1 Sergey V. Bondarchuk,3 Boris V.Afanasyev1

Hematopoietic stem cell transplantation and other therapeutic options in primary myelofibrosis: a review and two case reports

Elena V. Morozova, Maria V. Barabanshikova, Tatiana L. Gindina, V.V. Baykov, Boris V. Afanasyev

Allogeneic hematopoietic stem cell transplantation in children and adults with acute lymphoblastic leukemia

Sergey N. Bondarenko, Ivan S. Moiseev, Olga A. Slesarchuk, Elena I. Darskaya, Kirill A. Ekushev, Anna G. Smirnova, Alexander L. Alyanskiy, Tatyana L. Gindina, Elena V. Babenko, Elena V. Kuzmich, Elena V. Semenova, Alexander D. Kulagin, Luydmila S. Zubarovskaya, Boris V. Afanasyev

Correction of coronary endothelial dysfunction is a possible accessory mechanism for cellular therapy of the heart

Alexander S. Nemkov, Sergey. A. Belyi, Vladimir V. Komok, Oleg A. Grinenko, Nikolay S. Bunenkov

Experimental article

Evaluation of energy potential of fresh and stored bone marrow cells using a fluorescent potential-sensitive probe

Tatyana V.Parkhomenko, Oleg V.Galibin, Elena V.Verbitskaya, Vladimir V. Tomson

Clinical articles

Impact of initial serum ferritin on early post-HSCT complications: a single-center study

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Mostafa Shaheen,1,2 Maria O. Ivanova,1 Ivan S. Moiseev,1 Sergey V. Bondarchuk,3 Boris V.Afanasyev1

1. R.M. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, The First St. Petersburg Pavlov State Medical University, St. Petersburg, Russia
2. Hematology and Bone Marrow Transplantation Department, Tishreen Hospital, Damascus, Syria
3. Department of Hematology, Faculty Therapy, S.M.Kirov Military Medical Academy, St. Petersburg, Russia

Introduction

Iron overload (IO) is an important issue when treating patients who undergo hematopoietic stem cell transplantation (HCT). Elevated pre-transplant serum ferritin levels have been associated with increased morbidity and mortality after allogeneic HCT.
Patients and Methods In the single-center study, we have reviewed medical records of ninety-one consecutive patients (42 males and 49 females), with a median age at HCT of 31.6 years (range, 5 to 60), who underwent allo-HCT with unmanipulated grafts between Jan 2013 and Dec 2014.

Results

The median pre-HCT serum ferritin concentration was 765.35 (range, 12.1-4247) ng/mL for the total group. Fifty-three patients (58.24%) had initial serum ferritin of >500 ng/mL, and were assigned to the high-ferritin group. Increased pre-transplant ferritin concentrations were significantly associated with toxic or infectious complications of HCT, i.e., number of febrile neutropenic episodes (P=0.005), number of bacterial infection episodes (P=0.009), pneumonias (P=0.04), and demand for multiple RBC transfusions (P=0.04) within 100 days post-HCT. The significant association was found between pre-HCT ferritin concentrations (>773 ng/mL) and overall survival (P=0.04), disease-free survival (P=0.019), and mortality (P=0.02) among the groups. No significant relationships were observed between the initial ferritin levels and incidence of mucositis, or graftversus- host disease (P>0.05).

Conclusion

Measurement of serum ferritin, as a surrogate laboratory marker for IO, is quite practical for many hematological clinics. In the present study it was shown that the baseline increase of serum ferritin contents, is associated with higher risk of febrile episodes, infectious conditions, and slower recovery of myeloid cells, thus being of certain predictive value. Of special interest is an association between the pre-transplant ferritin levels and increasing demand for RBC transfusions after allo-HCT.

Clinical articles

Hematopoietic stem cell transplantation and other therapeutic options in primary myelofibrosis: a review and two case reports

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Elena V. Morozova, Maria V. Barabanshikova, Tatiana L. Gindina, V.V. Baykov, Boris V. Afanasyev

R.M. Gorbacheva Memorial Research Institiute of Children Oncology, Hematology and Transplantation, First St.Petersburg State I. Pavlov University, St.Petersburg, Russia

Primary myelofibrosis (PMF) is BCR/ABL–negative myeloproliferative disorder with splenomegaly, leukoerythroblasts, extramedullary hematopoiesis, reactive bone marrow fibrosis and neoangiogenesis with abnormal cytokine production, generally, affecting elderly persons. PMF may be accomplished by gradual bone marrow failure, splenomegaly, severe general (constitutional) signs, and clinical features of extramedullary hemopoiesis. Acute leukemia develops in up to 30% of PMF. A conventional cytostatic therapy of PMF does not affect the survival rates. Meanwhile, allogeneic hemopoietic stem cell transplantation (allo-HSCT) is the only curative procedure for the PMF.

PMF diagnosis is performed by appropriate WHO criteria and includes clinical, morphologic, cytogenetic, and molecular parameters. This disorder should be discerned from other myeloid neoplasias, i.e., polycythemia vera and essential thrombocythemia (ET). Differential diagnosis of PMF should also consider marrow fibrosis caused by other non-neoplastic or neoplastic conditions. In addition to JAK2 V617F or MPL mutations, a more recently found calreticulin (CALR) gene marker have been proposed for the MF (and essential patients with MF and ET).

Prognostic aspects are derived from a concept of PMF as a heterogeneous disorder with rather individual manifestation and evolution, with highly variable median survival of up to >20 years.

An appropriate scoring scale (IPSS) uses five risk factors to predict prognosis and stratify the patients into risk groups. The risk factors for leukemia-free survival include ≥3% circulating blasts, platelet count <100 x 109/L, and presence of unfavorable karyotype .

Drug therapy for PMF is not effective, in terms of overall or event-free survival. AlloHSCT for PMF is potentially curative, but still hazardous. Meanwhile, many patients may be observed for sufficient time without any therapy, whereas some of them are effectively managed by conventional treatment.

For low or intermediate-1 risk patients, there is no need for specific therapy in asymptomatic patients. Cytoreductive therapy may be reasonable in cases of extreme leuko- or thrombocytosis. The drug side effects include hepatotoxicity and virilizing effects for androgens, peripheral neuropathy (thalidomide), and myelosuppression (lenalidomide). The first-line therapy with hydroxyurea may reduce the spleen size in 40% of patients, with the spleen response lasting for ca. 1 year. Adverse effects of urea include myelosuppression and mucocutaneous ulcers.

Pegylated interferon is currently applied, early on in the course of ET or PV, with a goal of preventing or delaying fibrosis. The patients with intermediate-2 or high risk disease should be treated with investigational drugs, or alloHSCT. E.g., JAK1/JAK2 inhibitors (Ruxolitinib) are highly effective in PMF or ET MF. At the present time, ruxolitinib is the only FDA-approved JAK2 inhibitor for MF and the only modern non-HSCT therapy associated with increased survival. AlloHSCT is quite efficient, however, a hazardous intervention, thus being a method of choice for high-risk symptomatic younger patients with PMF. An evident effect of HSCT in MF is a quick restoration of normal trilineage hematopoiesis and functional microenvironment, with rapid and reversal of the myelofibrosis. In conclusion, considering the lack of long-term effective drug therapies for patients with MF, a potential risk of transplant-related complications seems to be proven in patients with DIPSS plus high- or intermediate 2–risk disease. JAK2 inhibitor provides a unique opportunity of implementing these novel agents into the transplant programs for high-risk patients. The general conclusions are illustrated by two clinical examples from authors’ experience showing successful outcomes of allo-HSCT in PMF.

Clinical articles

Allogeneic hematopoietic stem cell transplantation in children and adults with acute lymphoblastic leukemia

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Sergey N. Bondarenko, Ivan S. Moiseev, Olga A. Slesarchuk, Elena I. Darskaya, Kirill A. Ekushev, Anna G. Smirnova, Alexander L. Alyanskiy, Tatyana L. Gindina, Elena V. Babenko, Elena V. Kuzmich, Elena V. Semenova, Alexander D. Kulagin, Luydmila S. Zubarovskaya, Boris V. Afanasyev

Raisa Gorbacheva Memorial Research Institute for Pediatric Oncology, Hematology and Transplantation, First I.Pavlov State Medical University of St. Petersburg
The aim of this study was to evaluate efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in acute lymphoblastic leukemia (ALL), and to specify significant factors affecting clinical outcomes. Patients and methods. The study included 354 ALL patients aged 1 to 61 years who underwent allo-HSCT over a period of 1995 to 2015. Before HSCT, 24% of patients were in the 1st remission, 26% – in 2nd remission, 17%, in the ≥ 3rd remission; 34% of patients had active disease. Results. Overall survival (OS) was 47% when HSCT was performed in remission status versus 18% in patients transplanted in active disease state (p <.0001). Appropriate relapse incidence (RI) comprised 26% and 50%, respectively (P <.0001). Five-year OS was similar in children and adults (48% and 47% respectively, p>0.2). Pre-transplant remission state showed certain correlations with OS in pediatric and adult transplant patients, i.e., 79% vs 60% for HSCT in 1st remission; 40% vs 43% in 2nd remission, and 33% vs 23% for the patients treated in ≥ 3rd remission. ALL RI in children and adults were also comparable for HSCT carried out in 1st remission (21% vs 32%), 2nd remission (33% vs 17%), and 17% vs 23% for HSCT performed in ≥3rd remission (p>0.2). Most ALL patients underwent myeloablative conditioning regimen (MAC) before allo-HSCT (n=89). OS in MAC group was 53% versus 40% among patients who underwent reduced-intensity conditioning (RIC) regimens (n=70, p=0.04). The conditioning regimen intensity did not correlate with the RI after allo-HSCT (24% and 30% (MAC vs RIC respectively), p=0.09). Non-relapse mortality (NRM) did not significantly differ for children and adults (32% vs 37%, p>0.2), being dependent on the disease state: 21% vs 25% after HSCT in the 1st remission; 31% and 43%, when treated in the 2nd remission, and 50% vs 61% if transplanted in ≥3rd remission. Conclusion. Allo-HSCT from an HLA-matched related or unrelated donor is indicated in patients with high-risk ALL in first remission and in all the patients in the second remission.

Clinical articles

Correction of coronary endothelial dysfunction is a possible accessory mechanism for cellular therapy of the heart

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Alexander S. Nemkov, Sergey. A. Belyi, Vladimir V. Komok, Oleg A. Grinenko, Nikolay S. Bunenkov

Department of Cardiovascular Surgery, First St.Petersburg State I. Pavlov Medical University, St.Petersburg, Russia

Over 12 years of cellular therapy in cardiology, some dictinct positive results are obtained in randomized studies. However, exact mechanisms of hematopoietic stem cell actions are still unclear under these clinical conditions. Paracrine effects of cell therapy cannot explain all these effects. Enhanced neoangiogenesis upon stem cell injection is a proven mechanism for improvement of blood supply to the heart. Meanwhile, a decreased revascularization effect 3-4 years after cell therapy is followed by repeated myocardial improvement 6-9 mo after repeated cell infusions with active development of collateral vessels, thus suggesting an additional mechanism for improvement of coronary blood supply. Restoration of regulatory functions of endothelium and smooth muscle cells, including increased NO synthase activity of endothelium and its interactions with myocardiocytes may represent a probable mechanism for this action.

Experimental article

Mesenchymal stromal cells protect from consequences of HSCT-transplantation preparatory irradiation: insights into possible mechanisms

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Claudia Lange,1 Rudolph Reimer,2 Jozef Zustin,3 Bärbel Brunswig-Spickenheier1

1. Clinic for Stem Cell Transplantation, Dept. Cell and Gene Therapy, University Medical Center Hamburg-Eppendorf,
2. Technology Platform Microscopy & Image Analysis, Heinrich-Pette-Institut Hamburg,
3. Institute of Pathology, University Medical Center Hamburg-Eppendorf,

Abstract 

Ionizing irradiation is widely used as conditioning therapy in bone marrow (BM) transplantation. High-dose radiation treatment induces profound tissue damage, especially, of hematopoietic stem cells and progenitor cells. Efforts to improve clinical outcomes post- irradiation are focused on the hematopoietic stem cell niche. Mesenchymal stromal cells (MSCs) represent an integrative part of the BM stromal microenvironment. When co-transplanted with HSC, MSCs augment hematopoietic recovery after chemo- or radiotherapy. The aim of our study was to evaluate essential biological parameters of MSCs, with respect to their lineage-specific differentiation capacity, in vivo survival rates, as well as their ability to rescue lethally irradiated hosts. Materials and Methods. In vitro differentiation of human BM-derived MSCs (hMSCs) for hematopoietic (HSC) and endothelial cells (EC) was studied by reverse transcription- quantitative PCR (RT-qPCR) of lineage-specific surface markers and other proteins. To test in vivo ability of murine MSCs to rescue lethally irradiated (9.5 Gy) mice, the animals were transplanted with eGFP-marked murine MSCs (mMSCs). Long-term donor chimerism was assessed in blood, BM and thymus using CD45.2 and Y chromosome markers. A microarray analysis of bone marrow cells from MSC-transplanted animals was also performed, in order to compare their gene expression profiles to appropriate controls.

Results

Upon in vitro differentiation of hMSCs, the hematopoietically differentiated cells changed their gene expression towards a typical profile of progenitor and mature hematopoietic cells. A variety of transcription factors responsible for erythropoiesis, megakaryopoiesis, lympho- and myelopoiesis were up-regulated during differentiation in serum-containing media. A population of cells with small round or polymorphic nuclei was detected which expressed hematopoietic progenitor and mature antigen markers, albeit to a rather low degree. The same cells were able to acquire endothelial morphology and expressed endothelial genes upon cultivation with endothelial promoting factors. Following MSCs transplantation, the lethally irradiated mice showed normal hematopoietic recovery comparable to effects of HSC infusions. Seven months later, the recipients had normal distribution of peripheral blood cell populations. No evidence of donor chimerism was shown at any time

Experimental article

3D bioprinting applications for in vitro modeling of cellular interactions and tissues

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Joel C. Glover

Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo Norwegian Center for Stem Cell Research, Oslo University Hospital Professor, Institute of Basic Medical Sciences, University of Oslo Medical Faculty
The article considers different strategies for seeding stem cells and their progeny and construction of new tissues and organs, i.e., artificial biocompatible templates, natural decellularized templates, and generating complex tissues directly from stem cells and matrix materials using bioreactors or 3D-printing. Generally, culturing cells under 3D conditions allows to retain fully morphological and functional integrity of specialized cells as shown with hepatocytes. In particular, a promising approach to 3D organ fabrication is to use special bioprinters to prepare tissue scaffolds. Relevant studies have resulted in the production of organ-like cellular complexes, for example tubular/glomerular kidney structures. There are some technical issues which should be considered in any single case, including type of printing technology, choice of biomatrix type, printing parameters, etc.

Microextrusion tenchique and laser-induced forward transfer (LIFT) approach are considered as prospective printing technologies. Natural substrates for tissue and organ scaffolds could be obtained by decellularization and subsequent cell seeding, as already shown in animal experiments. Generating 3D tissue models could create promising opportunities for hematopoiesis research in its natural microenvironment.

Experimental article

Evaluation of energy potential of fresh and stored bone marrow cells using a fluorescent potential-sensitive probe

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Tatyana V.Parkhomenko, Oleg V.Galibin, Elena V.Verbitskaya, Vladimir V. Tomson

R.M. Gorbacheva Research Institute of Pediatric Oncology, Hematology and Transplantion, Research Center, First St.Petersburg
State I.P.Pavlov Medical University, Russian Ministry of Health Care, St.Petersburg, Russia

Bone marrow is a primary source of hematopoietic stem cells in clinical transplantation. Quality of bone marrow grafts is a key factor of their successful in vivo expansion. The aim of our work was to test a semi-quantitative technique for assessment of bone marrow cell viability under strict storage conditions, by means of a fluorescent membrane potential-sensitive 2-Di-1-ASP probe.
We have studied 20 samples of normal bone marrow cells (BMC). The cells were placed in a standard storage solution with sodium citrate, citric acid; phosphate salts, dextrose and adenine. Cell counts and viability tests were performed up to 72 hours of incubation. The samples were labeled with 2-Di-1-ASP probe at specified terms. Fluorescence intensity was measured for single nucleated cells, followed by calculating mean fluorescence values and myelokaryocyte numbers. Mitotic indexes were determined both in Giemsa-stained and fluorescent probe-stained cells. Cluster analysis and non-parametric tests were used for statistical evaluation.

Results

Initial cell survival of 80-92% was shown at 3…5 hours of storage, then decreased to 70-75% by the end of incubation. Meanwhile, cell incubation for 3 hours was accompanied by increased fluorescence, in terms of F̃ values, mainly, due to higher proportion of “bright” cell population (>100 arb.units, NF>100,%). D (ratio of NF>100 at 3h storage to NF>100 initial) proved to be the most informative parameter, thus enabling us to predict sample- specific differences for the F̃ values at later terms. All BMC samples exhibited increased F̃, on the account of brighter cell population (NF>100), over 3 hours of incubation. This increase correlated with increase in myelokaryocyte counts. An additional cluster analysis allowed us to classify the BMC samples into 3 sub-groups, by their significant inter-group differences for D values and cell number changes. In particular, a number of mitotic cells were detected in BMC populations at 5 to 24 hours of incubation, showing bright stainability with 2-Di-1- ASP probe. We revealed 0.60±0.10% of metaphase cells at initial time point. After 5-h storage, the frequency of mitotic cells increased to 1.4±0.1%; and, after 6-h colchicine treatment, the mitotic index increased to: 1.8±0.1%, thus showing good preservation of dividing cell fraction.
In summary, our results have shown sustained, and even increased energy activity using a potential-sensitive probe, and good survival of mitotic cell fraction under strict incubation conditions. Appropriate mechanistic studies of the bone marrow cell preservation and energy balance under the given storage conditions should be performed in future.