ISSN 1866-8836
Клеточная терапия и трансплантация

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Volume 5, Number 4
12/30/2016 02:22:00 pm
Volume 5, Number 4
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)
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

In the past few years there is a rising opinion in the scientific circles that the prospects of hematopoietic stem cell transplantation (HSCT) are diminishing due to approval of novel target and immunomodulating agents.

These targeted anticancer drugs have drawn attention of clinicians, scientists and social media. For instance, imatinib and other tyrosine kinase inhibitors (TKI’s) proved to be the first ‘magic’ bullet applied for treatment of chronic myeloid leukemia and other Ph-positive malignancies. Development of JAK-2 inhibitors (Ruxolitinib) paved further ways in therapy of some chronic myeloproliferative neoplasms.

A less specific approach includes eradication of malignant cell populations expressing a typical surface marker which could be attacked by the cytolytic drug. Clinical experience brings great expectations for Blinatumomab, an anti-CD-19/ CD3 bi-specific antibody applied for treatment of B-cell acute lymphoblastic leukemia (B-ALL). Hodgkin’s disease, another common disorder, is now more curable with cytolytic drug-antibody conjugate, Brentuximab, an anti-CD30+ monoclonal antibody.

The most encouraging results in clinical oncology have been quite recently obtained with the so-called immune checkpoint inhibitors which proved to be effective in a variety of solid tumors, as well as in hematological malignancies. Nivolumab, a PD-1 inhibitor, is the most advanced drug developed in the field. Specific action of these drugs on the target cells is based on blockage of a molecular checkpoint which regulates T-cell switch towards apoptosis. However, precise therapeutic effects of PD1-inhibitors should be further elucidated. In particular, we are only in the beginning of the path to understanding the mechanisms, facilitating the sensitivity and resistance to this group of agents. Therefore, several years are required in order to specify the place of PD-inhibitors in the treatment of different tumors.

Meanwhile, with accumulation of clinical data, it becomes clear that novel agents do not substitute HSCT, but rather significantly extend its curative potential through achievement of deeper remissions before the transplant and prevention of relapse as post-transplant therapy. This is particularly important for the patients resistant to conventional chemotherapy, and novel agents are applied as a ‘bridge’ to the subsequent hematopoietic transplantation. In this respect, further clinical studies are required for TKI’s, Blinatumumab, Brentuximab, immune checkpoint inhibitors, and other targeted drugs. On the other hand, there is emerging data that some of the target agents, like TKIs or Ruxolitinib, bear the significant potential for modulating graft-versus-host and graft-versus-leukemia effects, thus further improving the results of HSCT.

We are awaiting new reviews and original articles on the targeted drugs designed for treatment of oncological disorders. The CTT Journal is becoming popular among Russian- speaking specialists in the field of hematopoietic stem cell transplantation, due to broader coverage of different topics related to cellular therapy. The Journal is indexed in Scopus, thus increasing its visibility in the World Web. Moreover, the articles published in CTT are now adopted for Doctor’s Thesis by the Russian Higher Attestation Commission. We hope that the number of our authors and readership will also increase with time.

Clinical articles

Bloodstream infections and herpesvirus activation following intensive chemotherapy of adult oncohematological patients
Vitaly N. Chebotkevich, Stanislav S. Bessmeltsev, Ekaterina E. Kiseleva, Natalya P. Stizhak, Elena I. Kaytandzhan, Vitaly V. Burylev
Subclinical tubular epithelium damage and acute kidney injury following allogeneic hematopoietic stem cell transplantation
Vladimir A. Dobronravov 1,2, Kirill A. Smirnov 1,2, Boris V. Afanasiev 1,3, Olga V. Galkina 1,2, Alexey V. Smirnov 1,2

Experimental studies

Current methods and opportunities of next-generation sequencing (NGS) for HLA typing
1Youri A. Serov, 1Ildar M. Barkhatov, 1Anton S. Klimov, 2Andrey S. Berkos
Impact of recipient marrow stromal cells functional characteristics on engraftment after allo-HSCT
Ildar M. Barkhatov, Nikolay Yu Tsvetkov, Daniil E. Ershov, Marina Y. Lavrukhina, Alena I. Shakirova, Alisa Ya. Potter,
Anna G. Smirnova, Ludmila S. Zubarovskaya, Boris V. Afanasyev

Clinical articles

Bloodstream infections and herpesvirus activation following intensive chemotherapy of adult oncohematological patients

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Vitaly N. Chebotkevich, Stanislav S. Bessmeltsev, Ekaterina E. Kiseleva, Natalya P. Stizhak, Elena I. Kaytandzhan, Vitaly V. Burylev

Russian Research Institute of Hematology and Transfusiology, St. Petersburg; 2nd Sovetskaya St.16, 191024 St. Petersburg, Russia.

Professor Vitaly N. Chebotkevich, Russian Research Institute of Hematology and Transfusiology, St. Petersburg; 2nd Sovetskaya St. 16, 191024 St. Petersburg, Russia.

Phone: +7 (812) 717-29-58
Fax: +7 (812) 717-25-50
E-mail: vitnikcheb@mail.ru

Intensive cytostatic chemotherapy is a standard strategy for leukemia treatment. Meanwhile, such treatment causes negative effects, i.e., including lymphopenia, granulocytopenia and damage to tissue barriers associated with significant risks of infectious complications, especially, bacterial sepsis and viremia. Our study was aimed for identification of bacteremia and fungemia in oncohematological patients following intensive chemotherapy, and assessment of potential modifying role of herpesvirus infections.

We assessed frequency of infectious complications and their etiological agents in two groups of oncohematological patients treated at the Russian Institute of Hematology and Transfusion, especially focusing on mixed infections. Throat smears, venous blood, as well as urine and sputum specimens were taken for routine bacteriological cultures. Whole blood leukocytes were virologically tested by PCR, using standard examination protocol.

Our first virological study was performed for respiratory infections and included 85 randomly chosen patients. Influenza and parainfluenza viruses, respiratory syncytial virus, rhinovirus, adenovirus were detected in blood of single patients. Meanwhile, herpesviruses were detectable in 42% proportion of cases, i.e., HSV, EBV, and CMV DNA in blood cells were revealed in 5.2%, 26.3%, and 10.5%, respectively. Viral infections were not associated with positive bacteriological findings in this group. Our group of 33 clinical cases with proven sepsis observed among 64 patients. Generally, Gram-positive species prevailed over Gram-negative bacteria (69.2% versus 30.8%). However, the ratio of detectable Gram-negative flora was found to be increased from 23.1% to 40.2% between 2002 and 2013 (p<0.05). Coagulase- negative staphylococci (CoNS) prevailed among Gram-positive microorganisms, in particular, S. epidermidis and S. aureus), whereas Enterobacteriaceae, especially, E.coli, dominated among the Gram-negative bacteria.

Interestingly, 4 of 11 patients with coagulase-negative bacteremia had a concomitant herpesvirus infection: 2 cases were associated with EBV; 1, with CMV, and 1, with HHV6/EBV coinfection. Therefore, a high ratio of viral and bacterial co-infections may be revealed in a number of patients with proven sepsis. Moreover, common reactivation of herpesviruses may cause immunosuppression, or represent additional immunodeficiency markers predictive for bacterial infections at later terms. Therefore, one should take into account their predisposal for severe infectious complications when planning hematopoietic stem cell transplantation (HSCT) for these patients.

Clinical articles

Allogeneic transplantation of hematopoietic stem cells and solid organ in the same patient: an update

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Tapani Ruutu

Tapani Ruutu, Helsinki University Hospital, Biomedicum
2C, POB 705, FIN-00029 HUS, Helsinki, Finland

Phone: +358 50 555 1279
Fax: +358 9 4717 1506
E-mail: tapani.ruutu@hus.fi

Solid organ transplantation (SOT) is widely used for the treatment of end-stage organ insufficiency. Meanwhile, allogeneic hematopoietic stem cell transplantation (HSCT), by itself, causes frequent organ injury, thus being a potential indication for SOT. Due to increasing numbers of these interventions worldwide, a probability of combined disorders requiring both SOT and HSCT in the same patient is increasing. These transplantation combinations represent some clinical problems. Appropriate risk factors that may affect subsequent SOT outcomes post-HSCT include toxicity of HSCT procedure, immunosuppressive treatment for HSCT patients which may not be optimal for the SOT, potential problems caused by tissue incompatibility between host tissues, hematopoietic graft, and the solid organ graft, and emerging infections. Vice versa, in SOT recipients, the immunosuppressive medication may affect the organ functions, and increase the risk of infections. Moreover, SOT can be associated with development of hematological disorders, such as aplastic anemia, post-transplant lymphoproliferative disease, acute leukemias, etc. This paper aims at updating recent clinical experience with HSCT and SOT in the same patients. Here we discuss the survey which enrolled patients from 107 EBMT centres and clinical data on 45 SOTs carried out in the patients who previously underwent allo-HSCT. Kidney transplantations were performed, mainly, because of the drug-induced organ affection, or radiotherapy, whereas liver transplantations were made either early, for severe VOD or aGvHD, or later, due to chronic liver GVHD or cirrhosis. Survival rates and clinical outcomes are analyzed for each clinical situation in the groups. A special attention is given to the patients who received lung transplants in cases of bronchiolitis obliterans following allo-HSCT, characterized by inferior clinical outcomes and shorter survival time.

Some recent data concern allo-HSCT carried out after SOT. The hematological relapse rate was 22 %, thus allowing long-term observations. The 5-year rate for the solid organ failure was about 30%, mostly, due to graft rejection.

In summary, SOT can represent a valuable treatment strategy in HSCT recipients who develop an organ failure. Infections, graft rejection and other complications are frequent but usually manageable. Also, selected SOT recipients developing hematological disorders may benefit from allo-HSCT.

Clinical articles

Subclinical tubular epithelium damage and acute kidney injury following allogeneic hematopoietic stem cell transplantation

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Vladimir A. Dobronravov 1,2, Kirill A. Smirnov 1,2, Boris V. Afanasiev 1,3, Olga V. Galkina 1,2, Alexey V. Smirnov 1,2

Prof. Vladimir A. Dobronravov, MD, PhD, DSc, Research Institute of Nephrology, The First St. Petersburg I. Pavlov State
Medical University, Russia, L.Tolstoy St. 17, 197089, St. Petersburg, Russia

1First St. Petersburg I. Pavlov State Medical University, Russia,
2Nephrology Research Institute,
3Raisa Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, St. Petersburg, Russia

The aim of this study was to determine clinical value of molecular biomarkers (MBM), associated with tubular epithelium damage, for the prediction of acute kidney injury (AKI) in the setting of hematopoietic stem cell transplantation (HSCT).

Patients and methods

Ninety HSCT recipients (46 males, 44 females) were enrolled into the observational prospective study. Urinary concentrations of calbindin, clusterin, IL-18 (interleukin- 18), KIM-1 (kidney injury molecule-1), GST-π (glutathione S-transferase-π) and MCP-1 (monocyte chemoattractant protein-1) were measured in all patients before HSCT and at 5 consequent time points during early post-transplant period, along with routine clinical monitoring. AKI was defined according to the KDIGO (Kidney Disease Improving Global Outcomes) Guidelines.

Results

The incidence of AKI cases constantly increased during the observation period and reached the maximum level by the week 5 following HSCT. MBM elevation was observed more frequently than AKI and preceded the latter. Clusterin, MCP-1 and KIM-1 levels significantly correlated with subsequent serum creatinine values, measured a week after the MBM’s analysis according to multivariate linear regression models adjusted for other confounders. An increase in KIM-1 and/or MCP-1 urinary excretion was independently associated with a relative risk of AKI development. In summary, multiple renotoxic events early after HSCT commonly result in markedly increased urinary excretion of distinct molecular biomarkers, reflecting subclinical tubular injury in the absence of AKI criteria. The subsequent development of AKI can be predicted by means of KIM-1 and MCP-1 urinary excretion evaluation.

Clinical articles

Time course of immune recovery and viral reactivation following hematopoietic stem cell transplantation

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1Olga S. Pankratova, 2Alexei B. Chukhlovin

1Tampere University Hospital, Tampere, Finland
2R. Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, The St. Petersburg State I. Pavlov Medical University

Total depletion of innate and adaptive immune cell populations occurs after intensive chemotherapy and hematopoietic stem cell transplantation (HSCT) then followed by gradual recovery of immune populations, due to progenitors derived from donor hematopoietic cells which differentiate to myeloid and lymphoid lineages. Time dynamics of immune reconstitution and differential maturation of distinct immune populations is only partially evaluated, especially, at early terms post-transplant. E.g., innate immunity is restored within 1st month after HSCT, due to rapid reconstitution of granulocytes, monocytes, and natural killer (NK) cells. Meanwhile, functional recovery of mature NK-cell subsets and blood monocytes may continue for up to 3 months.

Both T- and B-lymphocyte pools are restored much slower than myelomonocytic populations. The available information on their post-HSCT immune recovery is limited, since most studies are performed at later terms (>1 month post-transplant). Absolute numbers of CD8+ T cells return to control values ca. 4 months post-HSCT, however, exhibiting a skewed repertoire of memory T lymphocytes. Recovery and maturation rates of CD4+ T cells largely depend on residual thymus function, especially, in young subjects. Hence, a naïve CD4+ T cell population in pediatric patients predominates over 6 months post-HSCT. In older persons with inactive thymopoiesis the total CD4 cell counts remain low for years after HSCT. Meanwhile, antiviral cellular immunity is active since early terms post-transplant. E.g., cytotoxic CD8+ cells specific for cytomegalovirus (CMV), or Epstein- Barr virus (EBV) rapidly expand in cases of CMV or EBV activation.

Despite recovery of absolute B-cell counts by day 30 post-HSCT, their functions, i.e., antigen-specific antibody production, are reduced for months and years after HSCT, due to slow restoration of mature immune cell populations, thus resemling normal evolution of B cell hierarchy in human organism.

Reactivation of herpesviruses (mostly, CMV, EBV and Herpes Simplex) is a known feature of immune deficiency. Timing of maximal herpesvirus incidence (2-3 months post-HSCT) corresponds to the period of CD8+ and CD4+ T cell functional deficiency and B cell immaturity, thus reflecting their suboptimal ability to eliminate herpesvirus-affected leukocytes. Individual terms of immune recovery after allo-HSCT depend on the patients’ age, source of donor cells, acute GvHD post-HSCT etc. Vaccination response, being a potent in vivo criterion of immune recovery in post-transplant patients, is also dependent on the subjects’ age and restored B cell functions. Time dynamics of specific antibody response shows that the patients with latent CMV reactivation may later exhibit a strong humoral immune response, thus making the infection self-limiting.

Clinical articles

Potential clinical applications of bone marrow-derived mesenchymal stem cells in bone regeneration

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Vladislav S. Sergeev

R. Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, The St. Petersburg State I. Pavlov Medical
University, St. Petersburg, Russia

Dr. V. S. Sergeev, St. Petersburg State I. Pavlov Medical University,
L. Tolstoy St. 6-8, 197022, St. Petersburg, Russia

Phone: +7 (911) 129 7580
E-mail: sergeev.vlad@gmail.com

Bone marrow (BM) contains a small resident cell population referred to as ‘multipotent mesenchymal stem cells’ (MSC). These adherent cells could be isolated and expanded in simple culture media and may differentiate in adipogenic or osteogenic pathway. So far an opportunity of MSC differentiation to hepatocytes, brain, or renal cells is not proven yet. Meanwhile, two potential clinical applications are considered for MSC: (1) as a tool for immune modulation in graft-versus-host disease (GVHD) and autoimmune diseases, or, (2) as a potential source of growth-promoting factors in specialized tissues. This heterogenous population may support hematopoiesis by secreting growth factors, cytokines and other biologically active substances. Upon injection, MSCs are able to migrate into damaged tissues, thus promoting their repair. However, only small MSC fraction may reach bone marrow niches following intravenous infusion. Multiple experiments with MSCs in different injury models show their ability to suppress apoptosis initiated by hypoxia, chemical agents/acidity and other deteriorating factors. This protective effect is mediated by a number of secreted growth factors, e.g., granulocyte-macrophage colony- stimulating growth factor (GM-CSF). A big number of clinical trials show high level of safety for the MSC therapy. Both clinical and experimental studies demonstrated only weak immunogenic effects of allogeneic MSC upon injection into immunocompetent recipients. At the present time, injections of in vitro expanded MSCs were performed in the patients developing acute GVHD after hematopoietic stem cell transplantation (HSCT), and in some autoimmune disorders.

Over last decade, several studies concerned potentially curative effects of MSCs injected into affected bone areas in the patients with osteogenesis imperfecta (OI), a severe inherited disease with altered collagen structure resulting into increased bone fragility. Here we present a synopsis of clinical protocol aimed for assessing safety, immunogenicity, and clinical effects of MSC injected to the OI patients during corrective osteotomy. One may suggest that a minor MSC subpopulation may migrate to the damaged areas differentiating to chondrocytes and osteoblasts, and, hence, contributing to the bone repair.

Experimental studies

Current methods and opportunities of next-generation sequencing (NGS) for HLA typing

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1Youri A. Serov, 1Ildar M. Barkhatov, 1Anton S. Klimov, 2Andrey S. Berkos

1Research Institute of Children Oncology, Hematology and Transplantation, The First State I. Pavlov Medical University,
St. Petersburg, Russia
2Russian Research Institute of Hematology and Transfusiology, Federal Medical-Biological Agency, St. Petersburg, Russia

Youri A. Serov, MD, Ph. D., CHS(ABHI), The Head of Biobank
Research Unit, Raisa Gorbacheva Memorial Institute of Children’s
Hematology, Oncology and Transplantation, The First
St. Petersburg I. P. Pavlov State Medical University, Leo Tolstoy
str. 6-8, 197022, St. Petersburg, Russia

Phone: +7(812) 338-6260 (office); +7(981) 973-8041 (mob.)
The region encoding human leukocyte antigens (HLA) is a 3.6-Mb segment at chromosome 6p21. A highly complex HLA system comprises important molecules involved in transplantation immunity, classical direct sequencing methods making it very complicating to resolve the genomic specificities of HLA genes, due of the complex nature of them. Current limitations of existing methods and the increasing rate of new alleles, strongly demanding for a new methodology approach for the HLA genotyping. Next-Generation Sequencing (NGS) is a method that can provide a complete solution to the HLA typing problem. Last decade, the development of (NGS) has shown the easy way for whole-genome analysis in individuals. HLA typing has also benefited from NGS technologies. Several high-throughput HLA- NGS-based typing methods were developed using different technological platforms. In this review, we summarize the possible applications and instrumentation developments of NGS in the in-depth HLA typing, with a focus on future clinical applications. However, some issues of bioinformatics and data mining remain an obstacle to more effective analysis of HLA analysis in the context of donor/recipient compatibility assessment.

Experimental studies

Lentiviral Hematopoietic Stem Cell Gene Therapy in Inherited Immune and Lysosomal Enzyme Deficiencies

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Gerard Wagemaker*

Erasmus University Rotterdam, The Netherlands; Center for Stem Cell Research and Development, Hacettepe University, Ankara, Turkey;
Raisa Gorbacheva Memorial Institute of Children Oncology, Hematology and Transplantation, St. Petersburg, Russia

Gerard Wagemaker, PhD, Professor, Erasmus University Rotterdam,
c/o PO Box 37048
3005 LA Rotterdam, The Netherlands

 Phone: +31-6-51619585
E-mail: g.wagemaker@genetherapy.nl
Rare diseases affect millions of people worldwide. Many of those are inherited disorders resulting in chronic disability and requiring cost-intensive care. Hematopoietic stem cell gene therapy has been developed over more than 20 years. At the state of the art, gene therapy is within reach for diseases in which (i) the genetic defect is identified, (ii) the diagnosis is made sufficiently early for a meaningful therapeutic intervention, (iii) a specific animal model is available for efficacy and safety evaluation. Appropriate therapeutic transgenes should also comply with certain biological criteria. Third-generation lentiviral vectors have been made self-inactivating (SIN) by deletion of enhancer regions from the LTR sequences thus reducing the risk of influencing nearby genes, resulting in favorable safety profiles. At the present time, lentiviral hematopoietic stem cell gene therapy has entered the stage of initial clinical implementation for immune deficiencies and lysosomal storage disorders. We discuss initial clinical trials using these vectors for selected metabolic storage disorders, which include adrenoleukodystrophy, metachromatic leukodystrophy, Hurler (MPS I), Pompe (GSD II), and Fabry diseases. This brief review summarizes the development and current clinical implementation of these approaches.

Experimental studies

Impact of recipient marrow stromal cells functional characteristics on engraftment after allo-HSCT

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Ildar M. Barkhatov, Nikolay Yu Tsvetkov, Daniil E. Ershov, Marina Y. Lavrukhina, Alena I. Shakirova, Alisa Ya. Potter,
Anna G. Smirnova, Ludmila S. Zubarovskaya, Boris V. Afanasyev

R. M. Gorbacheva Memorial Research Institute of Children Oncology, Hematology and Transplantation, and Chair of Hematology,
Transfusiology and Transplantation, The First St. Petersburg State I. Pavlov Medical University, St. Petersburg, Russian
Federation

Dr. Ildar M.Barkhatov, PhD, Laboratory Head, R. M. Gorbacheva
Memorial Research Institute of Children Oncology,
Hematology and Transplantation, The First St. Petersburg
State I. Pavlov Medical University, L.Tolstoy St. 6/8, 197022,
St. Petersburg, Russian Federation

Phone: +7(911) 778 2785
E-mail: i.barkhatov@gmail.com
Functional insufficiency of stem cell grafts is not rare in hematopoietic stem cell transplantation (HSCT), thus requiring further development of functional tests for recipient’s stromal cells, in order to substantiate clinical indications for co-transplantation of hematopoietic and stromal bone marrow cells. The aim of this study was to investigate a role of bone marrow stromal cells (BMSC) in the donor bone marrow engraftment, and their importance for development of complications after HSCT. Functional analysis of BMSC cultures from thirty-eight recipients of allogeneic hematopoietic stem cells has been shown that induced osteogenic differentiation correlated with faster hematopoiesis recovery in transplanted patients. Moreover, a pronounced ability for adipogenic differentiation proved to be associated with lower probability of the graft hypofunction. A pronounced hemostimulatory activity of stromal cells was observed in our study, thus suggesting effects of short-range hematopoietic growth factors from BMSC upon hematopoietic reconstitution. At the same time, a more pronounced pre-transplant expression of molecules which promote donor HSC homing by the recipient stromal cells was associated with faster hematopoietic recovery after transplantation.