Pediatr Transplantation 2013: 17: E189–E194

© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Pediatric Transplantation DOI: 10.1111/petr.12168

Chimerism of allogeneic mesenchymal cells in bone marrow, liver, and spleen after mesenchymal stem cells infusion Meleshko A, Prakharenia I, Kletski S, Isaikina Y. Chimerism of allogeneic mesenchymal cells in bone marrow, liver, and spleen after mesenchymal stem cells infusion. Abstract: Although an infusion of culture-expanded MSCs is applied in clinic to improve results of HSCs transplantation and for a treatment of musculoskeletal disorders, homing, and engraftment potential of culture-expanded MSC in humans is still obscure. We report two female patients who received allogeneic BM transplantation as a treatment of hematological diseases and a transplantation of MSCs from third-party male donors. Both patients died within one yr of infectious complications. Specimens of paraffin-embedded blocks of tissues from transplanted patients were taken. The aim of the study was to estimate possible homing and engraftment of allogeneic BM-derived MSCs in some tissues/organs of recipient. Sensitive real-time quantitative PCR analysis was applied with SRY gene as a target. MSC chimerism was found in BM, liver, and spleen of both patients. We conclude that sensitive RQ-PCR analysis is acceptable for low-level chimerism evaluation even in paraffin-embedded tissue specimens.

Alexander Meleshko1, Irina Prakharenia2, Semen Kletski3 and Yanina Isaikina4 1

Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus, 2 Clinical laboratory, Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus, 3Pediatric Pathology Department, Municipal Bureau of Clinical Pathology, Minsk, Belarus, 4Laboratory of Cellular Biotechnology and Cytotherapy, Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus Key words: mesenchymal stem cells – chimerism – mesenchymal stem cells homing – bone marrow transplantation Alexander N. Meleshko, 223053, Minsk region, Borovlany vil., Frunsenskaya srt, 43, Minsk, Belarus Tel./Fax: +375 (0) 17 265 42 22 E-mail: [email protected] Accepted for publication 5 September 2013

MSCs are adult stromal stem cells that can be isolated from BM and some other tissues. MSCs have a high potential for self-renewal and differentiation like all stem cells do. These cells are capable of differentiating into the connective tissue cells, such as fibroblasts, adipocytes, osteocytes and chondrocytes, as well as transdifferentiating into cells of other lineages, such as muscle, gut and lung epithelial cells, hepatocytes, and neurons. In the BM, stromal cells provide a scaffold, maintenance, and self-renewal of HSCs (1). Previous studies have shown that infusion of ex vivo expanded MSCs can improve the results of BM or HSCs transplantation in patients with cancer. The first clinical application of cultureexpanded autologous MSCs performed by Koc et al. (2) and Lazarus et al. (3). MSCs have low

Abbreviations: AML, acute myeloid leukemia; BM, bone marrow; GVHD, graft-versus-host disease; HSCs, hematopoietic stem cells; MNCs, mononuclear cells; MRD, minimal residual disease; MSCs, mesenchymal stem cells; SSR, simple sequence repeats; STR, short tandem repeats.

immunogenicity, and up to the present, both autologous and allogeneic human MSCs are safe and no adverse events or formation of ectopic tissues occurs (4), unlike murine MSCs that can give rise to sarcomas (5). Infusion of MSCs into patients has been shown to facilitate the engraftment of HSCs and reduce cytopenia (2, 6). Immunosuppressive capacities of MSCs promote alleviation of GVHD after allogeneic HSCs transplantation (4, 6–11). Although transplantation of HSCs normally results in full hematopoietic chimerism, data about engraftment of donor MSCs in BM and other tissues after transplantation are sparse and controversial. A number of studies demonstrated that after BM or peripheral blood HSCs transplantation, stromal cells of BM remained of host origin (12–15). After infusion of allogeneic culture-expanded MSCs, chimerism of BM stromal cells either was not detected (7), or was found in low level in small proportion of patients (16–18). In study of Ringden et al. (4), different tissues of three patients receiving MSCs infusions were observed for MSCs host–donor chimerism. In E189

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one of three patients, DNA of the MSC donor was detected in a lymph node and the colon, but not in the liver, spleen, and lungs. The chimerism level was estimated to be 10 5–10 6. In the other study by ten Hove et al. (19), liver chimerism was reported after allogeneic blood stem cell transplantation. These data indicate that if at all MSCs can engraft in some patients, these cells are capable of homing to different tissues and engraftment at a very low level. Therefore, reasonable method for expected MSCs chimerism detection must have sensitivity at least 10 3 and below. Conventional method of HSCs chimerism, PCR amplification of STR followed by fragment analysis, has the sensitivity limit at 10 2 (20). Several methods were applied for MSCs chimerism detection: fragment analysis of STR (7, 12, 15, 17), FISH with Y-chromosome-specific probes (12, 14), traditional PCR amplification of androgen and amelogenin genes (13), HLA genes (4), SSR polymorphisms (16), and real-time quantitative PCR (RQ-PCR) analysis of SRY gene (18). Among these methods, FISH and STRbased fragment analysis have sensitivity 1–5% which is not enough for expected level of MSCs engraftment. RQ-PCR is the most sensitive method for low-level target detection and allows reliable quantification and standardization as used for MRD detection in patients with leukemia (21). In our study, we investigated MSCs chimerism in paraffin-embedded blocks of the BM, liver, and spleen of two female patients died within one yr of infectious complications after allogeneic HSCs transplantation from female donors, who received infusion MSCs from third-party male donors. We applied highly sensitive RQ-PCR analysis with SRY gene as a target. Case report

Two patients received allogeneic BM transplantation as a treatment of hematological diseases and transplantation of MSCs from third-party donors. Our aim was to estimate possible homing and engraftment of allogeneic BM-derived MSCs in some tissues/organs of a recipient. Only paraffin-embedded tissue specimens from transplanted patients were available. Characteristics of both patients and donors are presented in Table 1. The patient 1, an eightyr-old girl with AML, received the haploidentical BM transplantation from her mother and infusion of MSCs simultaneously with allogeneic HSCs for enhanced hematopoietic engraftment. E190

Table 1. Clinical characteristics of patients and donors Characteristics

Patient 1

Patient 2

Patient age (yr) Patient gender Diagnosis

8 Female AML

Conditioning regimen

Treo36/Thio10/ Cyclo120/ATG60 Female Related/haploidentical Enhanced engraftment Male 1.6 9 106/kg Simultaneously

14 Female Diamond-Blackfan anemia Treo36/Cyclo120/ATG60 Female Unrelated/identical Acute GVHD (Grade III-IV) Male 1.2 9 106/kg Day 60



Fungal pneumonia


HSCs donors gender HSCs donors HLA type MSCs indication MSCs donors gender Dose of MSCs MSCs infusion postHSCs transplantation Death post-HSCs transplantation (days) Cause of death

Treo, treosulfan; Cyclo, cyclophosphamide; ATG, antithymocyte globulin; Thio, thiotepa.

The patient’s father was the MSCs donor. The patient did not reach full hematopoietic recovery, stayed cytopenic, and died from fungal pneumonia on the day 126 after BM transplantation. The patient 2, a 14-yr-old girl with DiamondBlackfan anemia, received MSCs on day 60 after allogeneic BM transplantation for treatment of acute gastrointestinal GVHD. The donor of HSCs for the patient was an unrelated woman, and MSCs were derived from BM of a thirdparty male donor. The girl died from infectious complications on day 177 after BM transplantation. The infusions of MSCs were performed after obtaining of informed consent approved by the ethical committee. MSCs were cultured ex vivo until the necessary amount had been obtained, depending on the body weight of each patient. Twenty to thirty days before planned allogeneic transplantation of HSCs, about 50 mL of BM aspirate was taken under local anesthesia from the posterior iliac crest of healthy donors. Methods MNCs were separated by Ficoll–Paque (Invitrogen, Carlsbad, CA, USA) gradient centrifugation, resuspended in Iscove’s modified Dulbecco’s medium (Invitrogen) with 10% FBS (Invitrogen), and transferred into 175 cm2 flasks filled with 30 mL medium, at a concentration of 2–3 9 106/ mL. The cells were incubated at 37 °C and 5% CO2. After reaching 80–90% confluence at the flask surface, the cells were detached with 0.25% trypsin–EDTA, and 106 cells were transferred into a new flask. Three passages were carried out in a similar way. After third passage cells were detached from the flasks surface with the trypsin, washed twice in Hanks’ solution, and resuspended in 20 mL of 0.9% NaCl solution. The ex vivo expanded cells were identified by flow cytometry as positive of typical MSCs surface

Chimerism of allogeneic mesenchymal cells markers (CD105, CD90, CD73) and negative of hematopoietic cells markers (CD34, CD45, CD14). MSCs suspension was infused intravenously. Paraffin-embedded blocks of BM, liver, and spleen were obtained after patients’ death from a pathology laboratory. Genomic DNA was extracted from paraffin by established method (22). Briefly, small pieces of tissue (2–3 mm3) were washed twice with 1 mL xylene, then twice washed with 1 mL ethanol, and resuspended in lysis buffer containing 0.5% SDS and proteinase K at the final concentration 0.7 lg/lL. After incubation at 55 °C for two to four h, DNA was isolated by standard phenol– chloroform extraction followed by ammonium acetate protein precipitation, and finally, DNA was precipitated by isopropanol. DNA pellet was washed by 70% ethanol, dried, and diluted in 50–200 lL of TE-buffer. DNA purity and concentration were determined by UV spectrophotometry (NanoDrop, Thermo Fisher Scientific) and diluted to 100 ng/lL. Control DNA from MNCs healthy male and female donors, DNA of recipients before transplantation and from MSC culture was extracted by similar method starting from lysis buffer. To avoid cross-contamination of samples, each sample was processed separately in different days. RQ-PCR analysis was performed on StepOneTM RealTime PCR System (Applied Biosystems, Foster City, CA, USA). SRY gene located at the Y-chromosome was used as a sex-related target for detection of MSC chimerism originating from male donor. Primers and probe were targeted to the 5′-part of the SRY gene, which is free of polymorphisms: forward primer 5′-AAGCCACACACTCAAGA ATGG-3′, reverse primer 5′-AGCTTTGTCCAGTGGCTG TAG-3′, TaqMan probe 5′-CACCAGCTAGGCCACTTA CC-3′. Design of primers and probe was performed with Primer3 online software (, and specificity was tested with NCBI BLAST. PCR amplification was performed in 20 lL reaction mix with QuantiTect Probe PCR Kits (Qiagen, Hilden, Germany), 5 lL (approx. 500 ng) of genomic DNA, 500 ng of each primer, and 150 ng of TM probe, labeled with 3′FAM, 5′BHQ1. For chimerism quantification, serial tenfold dilutions of male donor DNA in female DNA were used to make a standard curve construction. Chimerism can be quantified if the parameters of the standard curve are acceptable: correlation coefficient >0.95, slope between 3.1 and 3.9, and minimum range of three logs. All reactions including controls and tissue samples were set up in triplicates. For normalization, the same samples were amplified with primers to albumin control gene (23). An albumin standard curve was established using male DNA serially diluted in water. Standard quantity (SQ, mean of triplicate) was automatically generated by StepOneTM Real-Time PCR System based on standard curve for both albumin and SRY genes. Reduction (R) of follow-up sample was calculated as dividing SQ of male DNA by SQ of chimerism sample. Normalized reduction (NR) = R(albumin)/R(SRY). Reciprocal value 1/NR is chimerism level. As the expected level of MSC chimerism is very low, and DNA isolated from paraffin-embedded blocks is partially degraded, a very sensitive and specific method was required. We selected male-restricted gene SRY and RQ-PCR analysis with TaqMan probe as suitable method for this task. Our designed primers and probe combination was tested on DNA of blood samples of two male and eight female healthy donors. Both male donors showed perfect SRY amplification curves, and all female donors did not show

any amplification. Female DNA samples were pooled and used as a negative control DNA. Quantity and “amplificability” of DNA extracted from paraffin-embedded blocks was thoroughly tested by RQ-PCR amplification of reference gene albumin. In all cases, paraffin-derived DNA amplified at later cycles (Ct = 30–35), than fresh-cells DNA (Ct = 20–25) at the same concentration measured by spectrophotometer, due to modification of DNA during paraffin-block preparation. We achieved reproducible amplification of DNA with nice amplification shape and Ct in replicates

Chimerism of allogeneic mesenchymal cells in bone marrow, liver, and spleen after mesenchymal stem cells infusion.

Although an infusion of culture-expanded MSCs is applied in clinic to improve results of HSCs transplantation and for a treatment of musculoskeletal d...
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