Bone Marrow Transplantation (2003) 32, 947–952 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00 www.nature.com/bmt Microenvironment Constitution and telomere dynamics of bone marrow stromal cells in patients undergoing allogeneic bone marrow transplantation J-J Lee1,2, C-E Nam2, H Kook1, JP Maciejewski3, Y-K Kim1, I-J Chung1,2, K-S Park2, I-K Lee2, T-J Hwang1 and H-J Kim1,2 1Blood and Marrow Transplant Program, Chonnam National University Medical School, Gwangju, Korea; 2Genome Research Center for Hematopoietic Diseases, Chonnam National University Hospital, Gwangju, Korea; and 3Experimental Hematology and Hematopoiesis Section, Taussig Cancer Center, Cleveland Clinic Foundation, OH, USA Summary: cytotoxic chemotherapy or radiation, for example during the conditioning used forhematopoietic stem cell trans- We evaluated the genotypic origin of mesenchymal stem plantation (HSCT).3–8 This damage might result in impair- cells (MSC) following sex-mismatched allogeneic bone ment of hematopoiesis in BM following HSCT.9 Recently, marrow transplantation (BMT), and investigated the studies of stem cell plasticity in the field of HSCT have telomere dynamics in MSC in normal individuals and focused on the origin of MSC after HSCT and the patients after BMT. The study population consisted of 11 feasibility of stromal cell cotransplantation to enhance patients with hematologic disorders who showed complete engraftment of donor stem cells.9,10 Theoretically, BM chimerism after BMT. Telomere length was measured in grafts may also contain MSC and their engraftment would MSC using Southern blotting analysis in eight patients result in chimeric stromal elements. However, in most and 18 healthy subjects as a control group. Following reports to date, stromal cells were found to be of recipient culture, MSC were identified by the expression of SH2 origin.11–15 and SH4, and lack of CD14, CD34, and CD45. All MSC The telomeres are the terminal portions of eucaryotic showed the recipient genotype, based on the results of chromosomes composed of specific proteins/tandem DNA fluorescent in situ hybridization analysis using X-chromo- repeats, and are essential for the stability of chromosomes some satellite probes or microsatellite DNA polymor- and genes.16,17 Telomeric DNA in humans decreases by 50– phism analysis. The mean telomere length in MSC from 100 bp with each somatic cell division, and undergoes normal controls was 7.270.53 kb (range, 6.12–7.78), and progressive shortening with increasing age both in vitro and progressive telomere shortening was seen with age. There in vivo.18–20 Hematopoietic stem cells afterHSCT undergo was no significant difference in MSC telomere length increased replicative proliferation, resulting in further between the BMTgroup and age-matched controls. This acceleration of telomere shortening of the progenitor study confirmed that the MSC isolated from the recipients cells.21–24 MSC might also undergo excessive proliferative of allogeneic BMTdid not have the donor genotype, stress during reconstitution after HSCT. Thus, telomere despite complete chimerism. Moreover, MSC were length in MSC is expected to show accelerated shortening demonstrated to show progressive loss of telomere length following HSCT. However, the stromal microenvironment with age, but the telomeres in MSC were not affected by in BM has been reported to be severely damaged after BMT. HSCT, suggesting that decreased cycling may have no Bone Marrow Transplantation (2003) 32, 947–952. effect on telomere length.6,8 doi:10.1038/sj.bmt.1704253 In this study, we investigated the origin of MSC in Keywords: mesenchymal stem cell; telomere patients undergoing allogeneic bone marrow transplanta- tion (BMT). MSC cultured from BM of patients were confirmed to be derived from the host based on micro- satellite polymorphism determined by polymerase chain Adult bone marrow (BM) contains mesenchymal stem cells reaction (PCR) analysis or X-chromosome satellite DNA (MSC), which are capable of differentiation to osteoblasts, determined by fluorescent in situ hybridization (FISH) adipocytes, chondrocytes, myocytes, neural elements, and analysis, despite complete engraftment with donor-type stromal fibroblasts in vitro or in vivo.1–2 The stromal hematopoietic cells. In addition, we analyzed telomere compartment in the BM might be damaged by high-dose length in MSC from patients undergoing allogeneic BMT and normal controls by Southern blotting analysis. Telomere length in MSC cultured from normal control subjects showed progressive shortening with age, but Correspondence: Dr H-J Kim, Department of Internal Medicine, Chonnam National University Medical School, 8 Hakdong, Dongku, there were no significant differences in telomere length in Gwangju 501-757, South Korea; E-mail: [email protected] MSC between the BMT group and age-matched putative Received 23 March 2003; accepted 6 May 2003 controls. Constitution and telomere dynamics of stromal cells after BMT J-J Lee et al 948 Materials and methods Immunophenotypic analysis Patients To generate mouse monoclonal antibodies, human MSC hydridoma cells expressing SH2 or SH4 (1  107 cells) A total of 11 patients underwent sex-mismatched allogeneic (Osiris Therapeutics, Baltimore, MD, USA) were injected BMT between 1992 and 1998. The patients’ characteristics into the abdominal cavities of mice, and the ascites was are shown in Table 1. The patients ranged in age from collected and passed through a protein G-sepharose 6 to 36 years old, and the median period after BMT was column. After three passages, the presence of MSC was 3 years (range, 1–8 years). Engraftment was confirmed confirmed by flow cytometry with monoclonal antibodies by cytogenetic analysis, RBC phenotyping, FISH analysis to SH2, SH4, CD45, CD14, and CD34. CD14-fluorescein using an X-chromosome probe, and/or determination isothiocyanate (FITC), CD45-FITC, and CD34-FTIC of variable number of tandem repeats (VNTR) by antibodies were purchased from PharMingen (San Diego, PCR amplification. All patients studied were fully en- CA, USA). The MSC were incubated for 10 min in 1 ml of grafted, and were in complete remission at the time of 70% ethanol, and then washed with PBS containing 2% the study. BSA. The MSC were incubated with anti-SH2 or SH4 antibody for30 min at 4 1C, washed twice, and incubated with FITC-conjugated whole anti-mouse lgG (Sigma, St Normal controls Louis, MO, USA) for30 min at 4 1C. The samples were BM samples were collected from 18 healthy control examined with a FACSCaliburinstrument(Becton Dick- subjects, ranging in age from 4 to 74 years old, inson, San Jose, CA, USA), and the data were analyzed with normal blood counts. The normal control sub- using CellQuest software (Becton Dickinson). jects were healthy donors for allogeneic BMT and patients with lymphoma forstaging with normalBM Osteogenic differentiation of MSC morphology. To induce osteogenic differentiation, the MSC harvested after three passages were cultured with osteogenic medium MSC culture (0.1 mm dexamethasone, 10 mm b-glycerol phosphate, 50 mm l-ascorbic acid) for 11 days. The cells were fixed onto Mononuclear cells (MNCs) were isolated from all BM ProbeOn Plus microscope slides (Fisher Scientific, Pitts- samples from patients and normal control subjects by burgh, PA, USA) with 2% formaldehyde, and then Ficoll–Hypaque density gradient centrifugation. MNC developed with Fast Red TR salt (FRT) for10 min at were cultured in Dulbecco’s minimal essential medium room temperature to assess alkaline phosphatase activity in (GIBCO-BRL, Grand Island, NY, USA) supplemented osteoblasts. Counterstaining was performed with hematox- with 10% fetal bovine serum (GIBCO-BRL) at a density of ylin for 10 min at room temperature. 1  107 cells/30 ml in 175 cm2 polystyrene flasks. MSC were allowed to adhere for 72 h, followed by media changes FISH analysis every 3–4 days. When culture dishes reached confluence, adherent cells were passaged and replated at a density of To evaluate their chimeric status, the MSC on ProbeOn 1  106 cells/30 ml in 175 cm2 polystyrene flasks.5 After Plus slides were incubated with Pepsin solution for 5 min at three or more passages, the cells were harvested to perform 371C, and washed with PBS. The MSC were pretreated, morphologic, phenotypic, or functional studies and telo- then subjected to denaturation, hybridization, and ampli- mere length analysis. fication using the X-chromosome satellite probe (Vysis Table 1 Characteristics and engraftment kinetics for patients Case no. Diagnosis at transplant Age (years)/sex Conditioning regimen Time after transplant (years) Donor Chimerisma (%) 1 ALL 36/M TBI/CY 1 HLA-I sib 100 2 ALL 26/M TBI/CY 1 HLA-I sib 100 3 CML 25/F BU/CY 2 HLA-I sib 100 4 AML 9/M BU/CY 3 HLA-I sib 100 5 SAA 26/M CY/ATG/PCZ 8 HLA-I sib 100 6 SAA 10/M CY/ATG/PCZ 4 HLA-I sib 100 7 SAA 18/M CY/ATG/PCZ 4 HLA-I sib 100 8 SAA 26/F CY/ATG/PCZ 4 HLA-I sib 100 9 SAA 29/M CY/ATG 1 HLA-I sib 100 10 SAA 22/M CY/ATG/PCZ 4 HLA-I sib 100 11 SAA 6/M CY/ATG 2 HLA-I sib 100 ALL ¼ acute lymphoblastic leukemia; CML ¼ chronic myeloid leukemia; AML ¼ acute myeloid leukemia; SAA ¼ severe aplastic anemia; HLA-I sib ¼ HLA-identical sibling. Conditioning regimens for transplantation were: BU/CY ¼ busulfan, 4 mg/kg  4 days, cyclophosphamide, 60 mg/kg  2 days; CY/ATG7PCZ ¼ cyclophosphamide, 50 mg/kg  4 days, antithymocyte globulin, 30 mg/kg  3 days, procarbazine, 12.5 mg/day  3 days; TBI/CY ¼ 1200 cGy in eight fractions on 4 consecutive days, cyclophosphamide, 60 mg/kg  2 days. aChimerism expressed as percentage of donor cells using X-chromosome FISH analysis of 200 cells. Bone Marrow Transplantation Constitution and telomere dynamics of stromal cells after BMT J-J Lee et al 949 Inc., Downers Grove, IL, USA).