Microenvironment Constitution and Telomere Dynamics of Bone Marrow Stromal Cells in Patients Undergoing Allogeneic Bone Marrow Transplantation

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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 transplantation (BMT). MSC cultured from BM of patients were confirmed to be derived from the host based on microsatellite 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 Â 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 Â 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 Â 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 Â 4 days, cyclophosphamide, 60 mg/kg Â 2 days; CY/ATG7PCZ ¼ cyclophosphamide, 50 mg/kg Â 4 days, antithymocyte globulin, 30 mg/kg Â 3 days, procarbazine, 12.5 mg/day Â 3 days; TBI/CY ¼ 1200 cGy in eight fractions on 4 consecutive days, cyclophosphamide, 60 mg/kg Â 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). At least 200 nuclei were Statistical analysis examined in each sample by fluorescence microscopy. Values are presented as means7s.d. The Mann–Whitney U-test was used for comparison of telomere length PCR of minisatellite region D1S80 between groups. Linear regression analyses were performed to assess the correlation between telomere length The host or donor origin of MSC cultured from MNC of and age. BM was also determined by PCR amplification of the D1S80 locus, which contains a VNTR. Afterextractionof genomic DNA from MSC, PCR was performed using a DNA thermal cycler (GeneAmp PCR system 2400; Perkin- Results Elmer Cetus, Norwalk, CT, USA) according to the manufacturer’s protocol. Briefly, DNA was denatured for 2 min Generation and characteristics of MSC at 951C, followed by 30 cycles of amplification at 941C for MSC were successfully cultured from the BM MNC of 11 30 s, 651C at 40 s, and 721C for30 s. Afterthe last cycle, the patients and 18 normal control subjects, and in primary samples were held at 721C for10 min, and finally trans- cultures the MSC were seen to be spindle-shaped fibroblast- ferred to 41C. Electrophoresis of the amplified DNA was like cells (Figure 2a). Following seeding of 2 Â 107 MNC in carried out on 2% agarose gels. The amplification products primary cultures, the median MSC harvested was 6.2 Â 106 were visualized directly by staining with ethidium bromide. cells (range, 2.0–8.6 Â 106 cells) on day 24 (range, days 17–69). The yield of MSC in the patient group was not Measurement of telomere length significantly different from that in the normal control group. The immunophenotype study using flow cytometry Telomere length analyses were carried out as reported indicated that these cells (Figure 2a) expressed the previously.23,25 Briefly, all MSC obtained from eight mesenchymal-related antigens (SH2 and SH4), but not patients and 18 normal control subjects after three culture hematopoietic stem cell-related antigens (CD14, CD34, or passages were stored at À801C until DNA extraction. CD45). Cultured MSC showed a similar phenotype and Genomic DNA was extracted from the MNC using a expression of SH2 and SH4, but not CD14, CD34, or genomic DNA extraction kit (Bioneer, Choungwon, CD45. Chungbuk, Korea), and the integrity of extracted DNA was confirmed by 0.8% agarose gel electrophoresis. Differentiation of MSC to osteoblasts Aliquots of 4 mg of extracted DNA were completely digested with 40 U of RsaI, and the DNA was size To determine the differentiation capacities to multiple cell fractionated by electrophoresis on 0.7% agarose gels. types, the MSC were induced to generate osteoblasts in the DNA was transferred onto nylon membranes using the presence of osteogenic medium containing dexamethasone, capillary transfer method for Southern blotting. The filters b-glycerol phosphate, and l-ascorbic acid (Figure 2b). The were hybridized with a biotinylated telomere probe osteoblasts exhibited strong alkaline phosphatase activity, (TTAGGG)n (TeloQuant, Pharmingen, San Diego, CA, as demonstrated using FRT (Figure 2c). In addition, there USA) in hybridization buffer overnight at 651C. The were no differences in the osteogenic differentiation of hybridized probe was detected by the chemiluminescence MSC harvested after each subculture. method according to the manufacturer’s protocol (Telo- Quant). The telomere lengths were assessed quantitatively The marrow stroma remained of host origin after BMT by densitometric analysis of autoradiographs using a transmitter scanning video densitometer (Bio-rad, Segrate, Seven patients with aplastic anemia and fourpatients with Milano, Italy). The mean telomere length in each sample leukemia participated in this experiment. Owing to the was then identified as the peak intensity of the telomere long interval (1–8 years) after BMT, all patients were off length in kb by densitometry (Figure 1). immunosuppressive therapy and had been successfully engrafted, as confirmed by complete donor chimerism demonstrated by FISH analysis for X-chromosome and/or 9.4 PCR analysis to detect VNTR in separated DNA. 8.6 However, the MSC derived from BM MNC of the patients 7.2 showed FISH signals of recipients on analysis of at least 6.6 6.4 200 MSC using the X-chromosome microsatellite probe 5.7 (Table 2), and showed recipient-specific bands on analysis of VNTR (Figures 3 and 4). 4.4 4.8 4.3 Progressive telomere shortening in MSC of healthy normal Figure 1 Representative results of Southern blotting analysis of telomere controls length in MSC of transplantation patients and healthy normal controls. The vertical axis shows telomere length (kb). Lanes 1–6 and 7–10 show MSC were harvested from 18 healthy individuals after three DNA obtained from normal control subjects and patients of the following ages: 1, 4 years old; 2, 16 years old; 3, 22 years old; 4, 32 years old; 5, 42 culture passages, and telomere length was measured. The years old; 6, 52 years old; 7, 6 years old; 8, 25 years old; 9, 29 years old; 10, mean (7s.d.) telomere length in MSC from normal 36 years old, respectively. controls was 7.2070.53 kb (range, 6.12–7.78). This range

Bone Marrow Transplantation Constitution and telomere dynamics of stromal cells after BMT J-J Lee et al 950 a Table 2 FISH analysis for X-chromosome and mean telomere length in MSC of host afterBMT

Case Diagnosis Age Fish signalsa Mean no. at transplant (years)/sex telomere Recipient Donor length (kb)

1 ALL 36/M 210 (97.2%) 6 (2.8%) 7.69 2 ALL 26/M 233 (95.9%) 10 (4.1%) NM 3 CML 25/F 263 (98.9%) 3 (1.1%) 7.71 4 AML 9/M 387 (98.0%) 8 (2.0%) 7.78 5 SAA 26/M 261 (97.4%) 7 (2.6%) 7.42 6 SAA 10/M 242 (97.2%) 7 (2.8%) 7.96 7 SAA 18/M 218 (98.2%) 4 (1.8%) 6.23 8 SAA 26/F 248 (98.0%) 5 (2.0%) NM 9 SAA 29/M 286 (96.0%) 12 (4.0%) 6.88 10 SAA 22/M 235 (96.3%) 9 (3.7%) NM 11 SAA 6/M 252 (96.2%) 10 (3.8%) 7.57

aHealthy normal controls showed that the background level of other sex b signal was 0.49%.

Figure 3 FISH analysis of MSC using X-chromosome satellite probes showed one signal (left) and two signals (right) in male and female patients, respectively, following allogeneic BMT from sex-mismatched donors. c

PreBMT PostBMT Donor recipient recipient MNC MNC MSC

Figure 2 MSC in primary culture showing the morphology of spindle- Figure 4 Microsatellite DNA polymorphism analysis of DNA from shaped fibroblast-like cells (a). MSC (a) were tested for the ability to HLA-identical sibling donor(donorMNC) orrecipient patient MSC. differentiate in vitro to osteoblastic lineage cells (b). Osteogenesis (c)was Hematopoietic MNC DNA obtained from the recipient (patient MNC) visualized by the staining developed with FRT in alkaline phosphatase. was used as a control. The figure shows representative results of PCR amplification with D1S80 marker in patient #3.

of differences in telomere length was very narrow as Telomere length in MSC was not affected by BMT compared with those of MNC in healthy donors reported previously.23 Nevertheless, a progressive short- Telomere length of MSC from eight BMT patients (Table 2) ening of telomere length was detected in MSC with was measured after three culture passages. The mean age (P ¼ 0.001, r ¼À0.70). The telomere length in MSC telomere length (7s.d.) in MSC of the patients following of normal healthy control subjects was plotted by BMT was 7.4170.57 kb (range, 6.23–7.96), and there were the following equation: T ¼ 8.0–0.021 Â A, in which T is no signiﬁcant differences in telomere length of MSC the telomere length in kb and A is the age in years between the BMT group and age-matched putative controls (Figure 5). (7.5970.22 kb) (P ¼ 0.75).

Bone Marrow Transplantation Constitution and telomere dynamics of stromal cells after BMT J-J Lee et al 951 9 T= 8.0 - 0.021 × A clinical trials in progress using allogeneic MSC cotrans- (r = -0.70) plantation to improve engraftment and decrease GVHD in patients.32,33 8 Telomere length is maintained by a balance between replication rate and telomerase activity.18–20 The telomere length in primitive hematopoietic stem cells decreases despite telomerase activity. This ﬁnding explains that 7 telomerase activity in stem cells may be too weak to overcome the shortening of telomere length, especially after HSCT.21–24 However, variations have been reported in 6

Telomere length (kb) telomere length among healthy normal controls due to inherited heterogeneity.34 We found that MSC also showed progressive telomere shortening with age in healthy normal 5 controls. However, as compared with hematopoietic stem 0 1020304050607080 cells, the range of differences in telomere length in MSC according to age was very narrow.23 This finding suggests Age (years) that stromal cells constituting the microenvironment of BM Figure 5 Telomere length of MSC cultured from healthy normal control might require fewer doublings during the lifetime of an subjects plotted against age in years. The regression line is shown as a solid individual than hematopoietic stem cells. It has been line: T ¼ 8.0–0.021 Â A. P ¼ 0.001; r ¼À0.70; T ¼ telomere length (kb); A ¼ age (years). suggested that critically shortened telomeres in stem cells contribute to age-related clonal disorders,21–24 but the significance of the shortened telomeres in MSC remains unclear. Discussion In the present study, there was no significant difference in telomere length in MSC between the BMT group and age- The results of the present study confirm that the stromal matched putative controls. The hematopoietic stem cells cells of the BM microenvironment remain genotypically of after HSCT may be faced with increased replicative host origin after BMT, despite full hematopoietic engraft- proliferation and show accelerated telomere shortening, ment with donor-type cells. Although there have been some especially within 1 yearaftertransplantation. 21–24 However, controversial reports to the contrary, most recent studies the telomeres in MSC are not affected by HSCT. While the have indicated that stromal elements derived from donors reason for telomere stabilization is not clear, myeloablative do not contribute to reconstitution of the microenviron- conditioning regimens are known to damage stromal cells, ment afterHSCT, even afterlong-termfollow-up of as long and this may result in deficient proliferation of stromal as 27 years.11–15,26–29 The reason for this finding is not clear, cells.6,8 but possible explanations forthe lack of cells of donor In conclusion, ourfindings confirmthat the MSC origin include immune-mediated rejection of donor stromal cultured in BM MNC of recipients do not have the donor elements, inability to compete effectively with host stromal genotype. Moreover, MSC progressively lose telomere elements, and the low numbers of stromal cells trans- length with age, but the telomeres in MSC are not affected planted.9 In addition, a recent report suggested that the by stem cell transplantation. stromal compartment may be irreversibly damaged by the myeloablative regimens used in the preparation of stem cells for transplantation, and cannot be reconstituted by Acknowledgements committed stromal precursors.13 The feasibility of cotransplantation of MSC during This study was supported by a grant of the Korea Health 21 hematopoietic stem cell grafting is very interesting and is a R&D Project, Ministry of Health and Welfare, Republic of potentially medically important topic. For example, osteo- Korea (01-PJ10-PG6-01GN16-0005). genesis imperfecta patients showed 1.5–2.0% osteoblasts of donororiginfollowing allogeneic HSCT, suggesting a 15 potential therapeutic role of MSC in the field of HSCT. In References addition, MSC inhibit T-cell stimulatory capacity, as evidenced by inhibition of primary and secondary mixed 1 MajumdarMK, Thiede MA, Mosca JD et al. Phenotypic and lymphocyte reactions.30 This finding suggests that cotrans- functional comparison of cultures of marrow-derived mesen- plantation of MSC may prevent or ameliorate the chymal stem cells (MSCs) and stromal cells. J Cell Physiol development of GVHD following allogeneic HSCT.9 Koc 1998; 176: 57–66. et al 31 reported the results of a clinical trial using MSC in 2 Deans RJ, Moseley AB. Mesenchymal stem cells: biology and Exp Hematol advanced breast cancer patients who underwent autologous potential clinical uses. 2000; 28: 875–884. 3 Chamberlin W, Barone J, Kedo A, Fried W. Lack of recovery transplantation with peripheral blood. MSC infusions of murine hematopoietic stromal cells after irradiation-induced given at the time of autologous HSCT contributed to rapid damage. Blood 1974; 44: 385–392. hematopoietic recovery, enhancing neutrophil and platelet 4 Fried W, Chamberlin W, Kedo A, Barone J. Effects of engraftment, without any adverse effects as compared with radiation on hematopoietic stroma. Exp Hematol 1976; 4: those of historical controls. Currently, there are several 310–314.

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