The influence of autologous marrow mesenchymal stem cell infusion on hematopoiesis reconstitution after hematopoietic stem cells autotransplantation in children with oncological and hematological diseases
Yanina Isaikina, Nina Minakovskaya, Olga Aleinikova
Belarusian Research Centre for Pediatric Oncology and Hematology, Minsk, Belarus
Tel. +375 17 202 40 89, fax 202-42-22, e-mail: email@example.com
Accepted 19 May 2008
Published 28 May 2008
This investigation was undertaken to study the possibility for the application of mesenchymal stem cells (MSCs) for hematopoiesis support and reduction of the neutropenia period after autologous HSCs transplantation for children with oncohematological disorders and graft insufficiency of CD34+ cells/kg.
Patients and methods
24 children, who after collection of hematopoietic stem cells (HSCs) had low numbers of CD34+ (≤ 2,5 x106/kg) in autotransplant, were involved in our investigation. Autologous co-transplantation of MSCs was used for 7 adolescents; and 17 patients who were only given HSCs represented a control. The number of polychemotherapy cycles depended on the specific therapy response, relapse development, and the refractory to therapy, and varied from 4 to 10 cycles. MSCs were isolated from the bone marrow (BM) of patients up to 30–50 days before the autologous transplantation and expanded in vitro. CFU-F analysis was carried out for all patients. Statistical analysis was carried out with the help of STATISTICA 6.0 software. Difference reliability in groups during transplant parameters analysis was evaluated by the Mann-Whitney test, and the correlation degree between parameters by the Spearman test.
About 25 ± 6.9 ml of bone marrow was utilized in order to obtain MSCs. The CFU-F number was about 5.26 ± 0.6 colonies per 105 bone marrow mononuclear cells. The MSCs number had increased an average ~104 times after expansion in vitro for each patient. The data analysis revealed a statistically reliable dependence between the high-dose chemotherapy cycles number received by patients before bone marrow collection and MSCs growth time until a monolayer in primary culture (r = 0.79, p = 0.03) formed. The median number of MSCs reinfused into the patient was 0.6 (range 0.3–1.1) х 106 MSCs/kg in one hour after HSCs transplantation. In the case of co-transplantation, the MSCs neutrophil recovery > 500/µl was in 10 days (range, 9 to 11 days), ≥ 1000/µl in 11days (range, 10 to13 days) compared to 13 days (range, 11 to 15 days), and 14 days (range, 13 to19 days) respectively, in the control group (р = 0.002 and р = 0.001 correspondingly). A reticulocyte number of ≥ 1‰ was observed by 10 days (range 9 to 12 days) and 14 days (range 11 to 17 days), respectively (р = 0.004).
We determined an accelerated engraftment of HSCs transplant with low number CD34+ cells/kg in cases of autologous MSCs co-transplantation for children with malignant disorders. This approach is possible for children who have undergone prolonged myelotoxic and radiotherapy to expanse MSCs to efficient for co-transplantation volume.
Mesenchymal stem cells, colony forming unit-fibroblast, co-transplantation, hematopoietic engraftment
Generally, the treatment of children with advanced stages or relapses of malignant disorders consists of high-dose chemotherapy, or, sometimes, radiotherapy with follow-up autologous transplantation of hematopoietic stem cells (HSCs). Multiple courses of intensive chemo and/or radiotherapy result in damage to the bone marrow cells involving all natural factors of antimicrobial defense, resulting in the patients' susceptibility to a wide spectrum of opportunistic infections. During the early post-transplant period, these infections and appropriate therapeutic approaches are determined by neutropenia. After transplant engraftment, the risk of severe bacterial infections becomes greatly reduced.
For most patients, engraftment of peripheral blood stem cell (PBSC) autotransplant occurs relatively early: average neutrophil counts of > 500/μl are observed by 11 days; increase in platelets to > 20 000 is reached by 12 days. However, for about 5% of the patients transplanted with autologous HSCs, a delayed reconstitution of neutrophils and platelets is observed, which is explained mostly by a low dosage of transplanted CD34+ cells per kg of patient's body weight [1, 2].
A highly attractive method of choice to shorten the period of hematopoietic reconstitution after transplantation of HSCs is being developed at present, i.e., a simultaneous autotransplantation of mesenchymal stem cells (MSCs).
The main source of MSCs is bone marrow, which contains only 1-2 of these cells per 10000 of nucleated cells [3, 4, 5]. They have a fibroblast-like morphology and form specific fibroblast colony-forming units (CFU-F) when cultured in vitro. MSCs possess great proliferative capacity; and the cells obtained from young bone marrow donors are able to divide some 24-40 times, and to expand the cell population by up to 1 million times [6, 7, 8]. Phenotypic characterization of MSCs has shown that these cells are negative for hematopoietic markers (CD34, CD45, CD14, CD31, CD133), and positive for CD44, СD105 (SH2), CD166, CD73 (SH3), CD140 antigens [5, 9]. MSCs are multipotential progenitors and possess high plasticity. After cultivation in vitro and implantation, mesenchymal cells are able to differentiate into bone, cartilaginous, muscular, adipose tissues, and bone marrow stromal cells, as controlled by local factors and the microenvironment of the implantation area [5, 9, 10, 11, 12].
In such a way, MSCs give rise to bone marrow stromal cells that support hematopoiesis, by means of cytokine production (e.g., IL1, IL6, IL7, IL8, IL11, IL12, IL14), as well as Flt-3 ligand and SCF, which induces G-CSF and GM-CSF production [4, 9, 13]. Moreover, MSCs greatly support in vitro formation of megakaryocytes and platelet cells . This work concerns the issues of hematopoiesis reconstitution in children with oncohematological disorders. Autologous transplantation of HSCs has been recommended for these patients, but after HSCs mobilization and collection the amounts of CD34+ cells per kg were not sufficient for fast engraftment. Co-transplantation with MSCs was applied for these patients.
Patients and methods
Patients: Twenty-four children were involved in our investigation. All of them were initially included in the protocol of treatment in BRCPOH and had neoplastic disorders at an advanced stage (II-IV), i.e., Hodgkin’s disease (HD), non-Hodgkin's lymphomas and Ewing's sarcomas. Autologous transplantation of HSCs formed a part of the standard treatment protocols for these diseases. After collection of HSCs, we detected an insufficiency in all of these patients of CD34+ cells (≤ 2,5 x106/kg) in transplants, due to either weak mobilization response induced by G-CSF, or excessive weight. The study was performed over a relatively long time period (June 2003 to May 2005). After obtaining written consent from the patients or their parents, autologous transplantation of MSCs was applied to seven adolescents, in addition to PBSC autotransplantation (five patients) and bone marrow autotransplantation (two cases), to investigate the influence of MSCs upon the rates of autotransplant engraftment.
Eleven patients were auto-transplanted with PBSC (control group 1), and the six patients transplanted with bone marrow without MSCs autotransplantation were considered as control group 2.
Mobilization and collection of HSCs: PBSC mobilization in patients was carried out using G-CSF Granocyte (Sanofi – Aventis, France), according to the following schedule: G-CSF, 10 μg/kg within 24 hours subcutaneously for 5 to 10 days, depending on the body mass of the patients. The lymphocyte counts were observed on a daily basis. PBSCs were harvested when the lymphocyte numbers exceeded ≥ 25 х 106/ml. A blood cell separator "CS-3000+" (Baxter, USA) was used for stem cell apheresis.
The CD34+ cell numbers in the transplant were detected by flow cytometry (FACScan, Becton Dickinson, USA).
Mesenchymal stem cells: Up to 30–50 days before the planned PBSC auto-transplantation, 20–50ml portions of autologous bone marrow were taken under anesthesia via bone marrow puncture. Mononuclear cells were separated in Histopaque medium with a buoyant density of 1.077 (Sigma, USA), then twice washed in Hanks solution, re-suspended in Iscove’s Modified Dulbecco Medium (IMDM) with 10% FBS (Sigma, USA), and transferred into 175cm2 flasks filled with 30ml medium, at a concentration of 2-3 x 106/ml. The cells were incubated under 37оC at 5% СО2. Non-adherent cells were removed every 48 hours during the medium replacement. After reaching 80–90% confluence at the flask surface, the cells were detached with 0.25% trypsin-EDTA, and 1x10 cells were transferred into a new flask (passage 1). Several passages were carried out in a similar way. Thei in vitro expanded cells were identified by flow cytometry for the presence of typical MSC surface markers (CD105, CD90, CD140), and the absence of hematopoietic cells markers (CD34, CD45, CD14).
CFU-F analysis: To perform CFU-F counts, the mononuclear cells were isolated from bone marrow samples, and re-suspended in a full medium that contained IMDM supplemented with 15% FBS, L-glutamine, 2-mercaptoethanol and hydrocortisone (all reagents from Sigma, USA). Cell suspensions were then transferred into 60-mm Petri dishes. The cells were cultivated for 14 days (37оC, 5% СО2). After methanol fixation, the resulting colonies were stained according to Giemsa, and counted with inverted microscopy.
Statistical analysis: The data obtained was treated using STATISTICA 6.0 software. When analyzing the transplant parameters, the statistical significance of the differences between the groups was evaluated by the Mann-Whitney test. The degrees of correlations between the parameters were evaluated by the Spearman test.
In Table 1, we present the characteristics of seven patients involved in the experimental group who obtained additional infusions of MSCs. The age of these patients was 13 to 17 years; male/female ratio was 5:2. Two patients were diagnosed as Hodgkin’s disease (HD), stages II and IV; two patients suffered with non-Hodgkin B-type lymphomas, stage III and IV, two of patients had Ewing's sarcoma, and one had forward mediastinal germinoma, IV stage. Treatment of all the patients started in accordance with the protocols approved in BRCPOH (for Hodgkin’s disease, DAL HD 95, for Ewing’s sarcoma, ММCU-99, for Non-Hodgkin’s lymphoma, BFM-95 Rez, for germinoma, MAKEI-96). The numbers of chemotherapy cycles depended on the specific therapy response, relapse development, and the absence of a response to therapy; and varied from 4 to 10 cycles.
In addition to low CD34+ cell numbers in the transplant, the stabilization of the main disease in two weeks and Karnovsky Status >80% served as criteria for inclusion into this investigation. Two patients were in complete remission state, stabilization of the process was determined in two other patients, and there was no response to induction therapy in three adolescents.
In patients with Hodgkin’s disease, upon conditioning before autotransplantation of HSC, a high-dose polychemotherapy block BEAM was used (carmustin 300 mg/m2+ Ara-C 800 mg/m2+ etoposid 400 mg/m2+ меlphalan 140 mg/m2); in Ewing’s sarcoma, TioBuM-140 (tiophosphamide 600 mg/m2+ busulphan 16 mg/kg + меlphalan 140 mg/m2); with Non-Hodgkin’s lymphomas BuVPEnd (busulphan 480 mg/m2+ VP-16 900 mg/m2+ endoxan 4500 mg/m2), and in germinomas BuM-140 (busulphan 480 mg/m2+ меlphalan 140 mg/m2).
Table 2 shows the characteristics of the 17 patients involved in control group 1 (11 pts) and control group 2 (6 pts), who were subjected to autologous HSCT only.
Ex vivo MSCs expansion for autologous transplantation
For subsequent infusion together with autologous HSCs, the MSCs were cultured in vitro until the necessary amount had been obtained, depending on the body weight of each patient. Cells from passages 1 to 4 were used for autotransplantation in our patients. All mesenchymal cells obtained in vitro were morphologically homogeneous and exhibited a fibroblast-like shape in the monolayer at the surface of flasks. When re-suspended after trypsinization, they were large and rounded with a round nucleus, and they were three-fold larger than the neutrophils.
The main parameters of growth dynamics for MSCs obtained from the patients' mononuclear cells in vitro are listed in Table 3. About 25.7 ± 2.8 ml of the bone marrow was utilized in order to obtain the MSCs. Of this volume, we yielded as many as 486.43 ± 108.61 х 106 mononuclear cells. A 90% confluence cell layer was obtained in the primary culture at about 17 ± 2 days of incubation. The cells were then detached from the flasks' surface and counted (first passage). An average of 17.14 ± 4.38 х 106 of MSCs were obtained after cell growth in primary culture.
After bone marrow collection, CFU-F analysis was carried out for all patients. This test allowed us to detect the relative number of MSCs (proliferative cells active in culture) among the nucleated cell fraction, isolated from patients' bone marrow probes. The CFU-F number was about 5.26 ± 0.6 colonies per 105 bone marrow mononuclear cells.
Thus, the MSC number did increase ~103 times after incubation in the primary culture. After several passages were carried out, and the MSCs were incubated in subculture, their amount became ~104 times higher. Fig.1 represents the MSCs growth dynamics in subculture for each patient.
Fig. 2 reflects the correlation between the numbers of initial MSCs in the bone marrow able to proliferate and to form colonies in culture (CFU-F number), and MSCs number obtained from the primary culture before they underwent subcultures (r = 0.77; р = 0.04).
Data analysis revealed a statistically reliable dependence between the high-dose chemotherapy cycles number received by patients before bone marrow collection and the time taken for MSCs growth until a monolayer in primary culture was formed (r = 0,79, p = 0,03).
These results display the reduction of MSCs proliferative activity after chemo or radiotherapy.
HSCs and MSCs transplantation
Table 4 represents the HSCs and additional MSCs transplant characteristics for each patient. 5 children who had received PBSC as a transplant a median of 4.6 (range 3.0–6.9) х 108 /kg of nucleated cells were transfused including a median of 1.14 (range 0.64–1.3) х 106 /kg of CD34+ cells. Two patients who had received autologous bone marrow as a transplant 2.5 х 106 /kg and 8.1 х 106 /kg of nucleated cells were transfused including 1.0 х 106 /kg and 0.8 х 106 /kg of CD34+ cells, correspondingly. The median number of expanded MSCs reinfused into a patient was 0.6 (range 0.3–1.1) х 106 MSCs/kg in one hour after HSCs transplantation. Cells detached from the flasks' surface with the trypsine were washed twice in Hanks solution, counted and re-suspended in 20 ml of 0.9% NaCl solution. Intravenous injection of the cell suspension into the patient was performed within 5 minutes. The phenotype and sterility of the cell probes were analyzed.
Cells from passage 4 were used in 4 patients, cells from passage 2, in two patients. For one patient with IV stage HD, who had received 10 blocks of high-dose chemotherapy, MSCs from the first passage were used, in view of their weak proliferation activity, and, as a consequence, with prolonged growth in the main culture over 27 days, and attenuated cell proliferation after the first passage.
Hematopoietic engraftment after co-transplantation of HSCs and MSCs
Table 4 represents the results concerning the reconstitution of neutrophils and red blood cells after HSCs reinfusion for all patients. Five patients who received the MSCs infusion, in addition to autologous PBSC transplantation, and all patients in the control group had obtained 5 μg/kg of G-CSF, starting from the fifth day after transplantation until transplant engraftment. In the case of co-transplantation with MSCs, the median time of neutrophil recovery to ≥ 500/μl was on day 10 (range, 9 to 11), and ≥1000/μl was on day 11 (range, 10 to 13). These rates of neutrophil reconstitution are higher when compared to control group 1, where the median time of neutrophil recovery to ≥ 500/μl was on day 13 (range, 11 to 15), ≥ 1000/μl was on day 14 (range, 13 to 19).
Reconstitution of red blood cells was determined by the appearance of ≥ 1‰ of reticulocytes in the peripheral blood. This value was also higher in the experimental group with MSCs infusion compared to control group 1, and median red cells recovery was on day 10 (range 9 to 12) and on day 14 (range, 11 to 17) respectively.
In two children with co-transplantation of MSCs and autologous bone marrow, neutrophil recovery ≥ 500/μl in peripheral blood analysis was detected for both patients on day 14, ≥ 1000/μl on day 16 and day 14, the reticulocytes number ≥ 1‰ was on day 16 and on day 14, correspondingly. These rates of reconstitution are higher when compared to the autologous bone marrow transplantation in control group 2 (without MSCs infusion), where a median neutrophil recovery ≥ 500/μl was observed on day 24 (range 18 to 32), ≥ 1000/μl on day 25 (range 20 to 35) and median reticulocytes number ≥ 1‰ recovery was observed on day 26 (range 17 to 28).
In our present work, we have for the first time proposed the viability of the curative potential for MSCs obtained from the bone marrow of children with oncohematological disorders, who have been pre-treated with high-dose polychemotherapy and radiotherapy. This investigation was undertaken in order to design a therapeutic strategy of MSCs application for hematopoietic support, and the reduction of the neutropenic period after an autologous transplantation of HSCs in children with insufficient amounts of CD34+ cells/kg.
The biological aspects of mesenchymal stem cells are still under intensive investigation. In spite of scarce experimental data, bone marrow-derived MSCs are increasingly being used in clinical settings. In most cases, either MSCs from healthy donors are cultured and expanded for use in allogenic co-transplantations with HSCs, or autologous MSCs are employed as implants for treatment of some non-oncological conditions, e.g., bone or cartilaginous disorders.
MSC's ability to support hematopoiesis in vitro has been shown experimentally by many workers, when co-culturing MSCs and HSCs. Mesenchymal stem cells are considered the precursors of stromal stem cells, osteoblasts, adipocytes, and endothelial cells that form areas of hematopoiesis-inducing microenvironment in bone marrow, thus supporting production of leucocytes, red blood cells and platelets [15, 16]. On one hand their effects upon hematopoietic precursors are exerted via secretion of soluble cytokines, chemokines, peptides, mediators and hormones and, on other hand, by formation of extracellular matrix from collagen, fibronectin and laminin molecules that provide homing and adhesion of hematopoietic cells. Interleukin-6 (IL-6), IL-1, IL-7, IL-8, SCF, Flt-3-ligand, colony-stimulating factors (CSFs), thrombopoietin, insulin-like growth factor, and transforming growth factor (TGF) are permanently synthesized by stromal cells, thus supporting the stability of blood cell counts within steady limits. IL-1 is the main inducer of cytokine production, whereas TGF is able to inhibit hematopoiesis .
Some studies concerning the capacity of cultivated MSCs to support human hematopoiesis in vitro have revealed that, on contact with MSCs, both primitive hematopoietic precursors and committed cells are able to proliferate, thus maintaining their ability for self-replication and differentiation in renewing hematopoietic tissues . The role of stromal cells is especially important due to their ability to prevent HSCs apoptosis [19, 20].
Graça Almeida-Porada et al, in their work on the influence of in vitro cultivated stromal cells on HSCs engraftment, used a xenogeneic model of prenatal transplantation of human cells into sheep embryos. They showed that in cases of human HSCs and MSCs co-transplantation, earlier and higher levels of donor cells in blood were attained. Moreover, HSCs engraftment was more effective with a combined treatment than HSCs transplantation alone . Investigations of human hematopoietic cells from umbilical blood and MSCs co-transplantation into NOD/SCID mice confirmed the importance of MSCs in fast and stable hematopoietic cell engraftment decisively .
At the present time, limited data has been published concerning the application of hematopoietic and mesenchymal cell co-transplantation in clinics in order to shorten the period of neutropenia post-transplant in patients with malignant disorders. To our knowledge, no publications exist that concern the results of autologous MSCs utilization aiming to support fast and prolonged engraftment of HSCs auto-transplants in children with oncological and hematological diseases.
The issues of delayed reconstitution of granulocytes and platelets after HSCs autotransplantation still exist, however. A problem with autotransplant rejection still remains, and, in most cases, it occurs due to low doses of transplanted CD34+ cells/kg [1, 2]. At present, all studies on the application of autologous HSCs and MSCs co-transplantation within different groups of patients deal with high doses of CD34+ cells/kg in transplants: e.g., patients with breast tumors received an average of 13.9 х 106 CD34+ cells/kg, according to Кос О. et al (2000). For related allogenic PBSC transplantation to patients with malignant hematological diseases Lazarus HM et al (2005) employed a mean 5.0х106 CD34+ cells/kg . And in the case of HSCs allogenic transplantation following T-cell depletion, D Cilloni et al (2000) transfused ≥ 2.2 х 106 CD34+ cells/kg . However, numerous observations in large cohorts of patients with PBSC autotransplants suggest high levels of CD34+ in the transplant to be among the most important factors that affect neutrophil recovery after prescribed myeloablative therapy. A standard threshold dose of CD34+cells/kg weight for engraftment of autotransplant and hematopoiesis reconstitution is ≥ 2.5 х 106 cells/kg [25, 26, 27], and, at infusion doses over 5 х 106 CD34+cells/kg, the duration of cytopenia is noticeably reduced [28, 29, 30].
The main idea of our pilot study was to demonstrate the efficacy of supplementary autologous MSCs transplantation, in order to accelerate hematopoietic stem cells engraftment at low doses of CD34+ cells in transplants obtained by leukapheresis: i.e., 0.64–1.3 х 106 CD34+ cells/kg in PBSC autotransplant, and 0.8–1.0 х 106 CD34+ cells/kg in bone marrow autotransplants. An analysis of the resulting data revealed a significant reduction in the post-transplantation cytopenia period for the patients with MSCs co-transplantation after sub-optimal CD34+ cells mobilization, when compared to the control group. In particular, neutrophil reconstitution ≥ 500/μl was found at 9–11 days compared to 11–15, platelet increase ≥ 10000/μl, in 10–13 days as compared to 13–19 in controls, and red cells increased at 9–12 days against 11–17 days in the control group. In this case, it enabled us to avoid the commonly accepted procedure of repeated attempts at PBSC collection, or harvesting considerable bone marrow volumes in case of insufficient CD34+ cell numbers in grafts upon primary harvesting. This is important for the patients who have undergone multiple cycles of chemotherapy or high-dose radiotherapy before autotransplantation, because the negative influence of chemo and radiotherapy on CD34+ cell number is well documented. Bensinger et al (1994) have revealed that each cycle of chemotherapy results in reduction of CD34+ cell numbers by 0.2 х 106 cells during leukapheresis for patients without radiotherapy. Meanwhile, a round of radiotherapy results in CD34+ cells number reduction by 1,8 х 106 after PBSC collection .
A number of publications have demonstrated that bone marrow stroma suffers significantly as a result of high-dose chemotherapy or radiotherapy [31,32]. Stromal cells (4–5 week cultures) from the patients after conditioning with busulphan and cyclophosphamide are able to produce monolayers in only 20% of cases, in comparison with 80% in healthy donors , and it is not always possible to obtain enough MSCs in vitro for infusion into patients .
Our data also confirms a reduced MSCs proliferation capacity after chemotherapy or radiotherapy in our patients. This trend is supported by high correlation coefficient (r = 0.79, p = 0.03) between the numbers of previously received high-dose chemotherapy cycles, and MSCs expansion rate.
In our work we evaluated the functional state of stromal cells by their ability to proliferate in children with supplementary MSCs transplantation. To this purpose, CFU-F analysis was performed. The average CFU-F numbers in bone marrow of the patients was 5.26 ± 0.52 per 105 mononuclear cells. This number was considerably lower when compared to the results of CFU-F analysis in healthy donors . In spite of this, the application of this MSC isolation method from bone marrow, and the technology of cell expansion considered by Кос О., we expanded the primary amounts of MSCs by ~104 times. Thus we obtained sufficient amounts of MSCs within a mean of 36,6 ± 6,3 days after bone marrow aspiration.
Moreover, analysis of our data shows that MSCs infusion is already efficient at MSCs dose 0.3 х 106 cells/kg, thus being quite important in cases of MSCs expansion at limited times elapsing from MSCs aspiration to co-transplantation.
As a result of our pilot study, we confirmed that autologous MSCs co-transplantation may accelerate engraftment of HSCs transplants with low numbers of CD34+ cells/kg when treating children with malignancies. Expansion of MSC in sufficient amounts is an acceptable option in auto-transplants for children after they received prolonged myelotoxic therapy and radiotherapy.
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Accepted 19 May 2008
Published 28 May 2008