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First published online July 12, 2007
Stem Cells Vol. 25 No. 10 October 2007, pp. 2670 -2676
doi:10.1634/stemcells.2007-0269; www.StemCells.com
© 2007 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Mitochondrial DNA Sequence Heterogeneity of Single CD34+ Cells After Nonmyeloablative Allogeneic Stem Cell Transplantation

Yong-Gang Yaoa, Richard W. Childsa, Sachiko Kajigayaa, J. Philip McCoy, Jr.b, Neal S. Younga

aHematology Branch and
bFlow Cytometry Core Facility, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland, USA

Key Words. Single-cell analysis • Mitochondrial DNA • Nonmyeloablative allogeneic stem cell transplantation • CD34+ cell • Kinetics

Correspondence: Yong-Gang Yao, Ph.D., Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Building 10 CRC, Room 3E-5140, 10 Center Drive, Bethesda, Maryland, 20892-1202, USA. Telephone: 301-451-7151; Fax: 301-496-8396; e-mail: yaoy3{at}nhlbi.nih.gov or e-mail: ygyaozh{at}yahoo.com

Received on April 12, 2007; accepted for publication on July 2, 2007.

First published online in STEM CELLS EXPRESS  July 12, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
We applied a single-cell method to detect mitochondrial DNA (mtDNA) mutations to evaluate the reconstitution of hematopoietic stem cells (HSCs) and committed progenitor cells after nonmyeloablative allogeneic stem cell transplantation in humans. In a total of 1,958 single CD34+ cells from six human leukocyte antigen-matched sibling donor and recipient pairs, individual CD34+ clones were recognized based on the observed donor- or recipient-specific mtDNA sequence somatic alteration. There was no overall reduction of mtDNA heterogeneity among CD34+ cells from the recipient after transplantation. Samples collected from two donors over time showed the persistence of certain CD34+ clones marked by specific mutations. Our results demonstrate the feasibility of distinguishing donor and recipient individual CD34+ clones based on mtDNA mutations during engraftment. HSCs were not limited in number, and similar mtDNA heterogeneity levels suggested representation of the total stem cell compartment during rapid hematopoietic reconstitution in the recipient.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Hematopoiesis is a hierarchical system, founded on primitive and generally mitotically quiescent hematopoietic stem cells (HSCs) that undergo either self-renewal or differentiation to multipotential and then committed progenitor cells, followed by differentiation into mature but proliferatively terminal circulating blood cells [1–3]. Understanding of the mechanism and kinetics of human HSC mobilization, homing, and repopulation has been informed by animal models [4, 5]. Long-term, multilineage hematopoietic reconstitution can be restored in lethally irradiated mice by a few HSCs or even a single HSC, with donor-derived chimerism being detected for prolonged periods after transplant [6–9]. However, in larger animals, such as cats, under homeostatic conditions, the total number of HSCs actively contributing to the production of blood cells is both conserved and stable over time [10–13]. Human studies suggest a similar physiology [14] but are limited because of the absence of a general method of distinguishing individual progenitor cells or their progeny.

Nonmyeloablative allogeneic stem cell transplantation (NASCT) has been explored as a safer alternative than conventional myeloablative transplantation for patients with a poor performance status, of advanced age, or with hematological malignancies and solid tumors refractory to standard treatment [15–22]. Biologically, the procedure has interest because low-intensity transplant preparative regimens produce a state of mixed chimerism, in which lymphohematopoietic cells of both the donor and recipient can be detected soon after engraftment. Engrafting donor T cells mediate beneficial and potentially curative graft-versus-malignancy effects, and they are also responsible for the more gradual eradication of recipient marrow by immune elimination; although engraftment kinetics vary among patients, chimerism may ultimately convert from mixed to full donor in origin in both myeloid and lymphoid lineages [23, 24]. Complete donor T-cell chimerism is desirable as it enhances the alloreactivity believed to underlie disease eradication and to prevent relapse [16, 17, 22, 25, 26]. Although there are data on engraftment kinetics of mature cells such as myeloid, B-cell, and T-cell populations [22], the dynamics of donor HSC engraftment and differentiation in the recipient following NASCT has not yet been characterized.

Recently, we established a method to analyze mitochondrial DNA (mtDNA) sequence variation of single cells that are present in the bone marrow and peripheral blood [27–30]. Our original studies were directed to the possible role of mtDNA mutations in aging and hematologic diseases. However, and surprisingly, we observed considerable variability among individual CD34+ cells and later among lymphocytes and granulocytes, as well as in leukemia blast cells, despite assessing sequence variation in only a small portion of total mtDNA [27, 28, 30, 31]. Some small clones, defined by mtDNA-specific somatic mutation(s), appeared to be derived from a true HSC, as the same variant was observed concurrently in different cell lineages [28]. These studies suggested the feasibility of using mtDNA variation as a natural marker to monitor HSC number and activity in healthy donors [28] and the dynamics of leukemia blasts [30].

In the current work, we sought to characterize donor- and recipient-specific mtDNA mutations in single CD34+ cells to track donor- or recipient-specific clones during and following engraftment. In addition, the comparison of mtDNA variation from sibling samples and the same donor over time might provide information regarding the mtDNA mutation pattern in maternally related individuals and longitudinal mtDNA alterations in single CD34+ cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Patients and Transplant Procedure
We collected peripheral blood (PB) from six patients (one with aplastic anemia, one with pancreatic cancer, one with gastric cancer, and three with renal cell cancer) before and after NASCT and obtained blood also from their human leukocyte antigen (HLA)-matched sibling donors (Table 1). Except for the gastric cancer patient/donor sample pair, the sibling pairs were sex-matched. Patients and their donors gave informed consent, and the nonmyeloablative allogeneic stem cell transplantation was performed on sequential protocols [16, 17, 32] approved by the institutional review board of the National Heart, Lung, and Blood Institute. Our nonmyeloablative, reduced-intensity conditioning regimen was as follows: 60 mg/kg cyclophosphamide on days –7 and –6 and 25 mg/kg fludarabine intravenously on days –5 to –1; from day –4, cyclosporine was given intravenously at 3 mg/kg or orally (twice daily) at 5 mg/kg when tolerated, both to maintain blood levels in the therapeutic range. Donors received 10 µg/kg granulocyte colony-stimulating factor daily for 5–6 days to mobilize CD34+ cells; mobilized peripheral blood HSCs were collected by leukapheresis on day 5 and on days 6 and 7 (if necessary) to achieve a target dose (>3 x 106 CD34+ cell/kg recipient weight transplanted in all cases). More details of the procedure for patient CABL have been described [32].


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  		Table 1. Sample information and mitochondrial DNA mutation levels in single CD34+ cells

 
Single-Cell Sorting, Amplification, and Sequencing
Mononuclear cells from heparinized PB were separated by Ficoll density gradient centrifugation and stored frozen in liquid nitrogen until they were subjected to sorting. We followed the staining method for anti-CD34 phycoerythrin-conjugated monoclonal antibody (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) provided by the manufacturer and the procedure described in our recent studies [28, 30]. Single CD34+ cells were sorted into a 96-well plate at a mode of one cell per well on a MoFlo Cytometer (DakoCytomation, Fort Collins, CO, http://www.dakocytomation.com) and were digested in 50 µl of lysis buffer per well (10 mM Tris-HCl [pH 8.0], 50 mM KCl, 100 µg/ml proteinase K, and 1% Triton X-100) at 56°C for 20 minutes, followed by an inactivation of the proteinase K at 96°C for 8 minutes.

Two-step nested polymerase chain reaction (PCR) amplification was performed under the same conditions described previously [30]. In brief, the first PCR was performed in 30 µl of reaction mixture containing 400 µM of each dNTP, 1 unit of TaKaRa LA Taq (with proofreading activity; Takara, Otsu, Japan, http://www.takara.co.jp), 0.5 µM each primer (L15594: 5'-CGCCTACACAATTCTCCGATC-3'/H901: 5'-ACTTGGGTTAATCGTGTGACC-3'), and 5 µl of cell lysate on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com). The amplification procedure includes one denaturation cycle of 94°C for 3 minutes; 35 amplification cycles of 94°C for 30 seconds, 52°C for 40 seconds, and 72°C for 1 minute with a 5-second increase per cycle; and a full extension cycle of 72°C for 10 minutes. The second PCR was performed in 50 µl of reaction volume containing 400 µM of each dNTP, 2 units of LA Taq, 0.5 µM of each primer (L15990, 5'-TTAACTCCACCATTAGCACC-3'; H650, 5'-GAAAGGCTAGGACCAAACCTA-3'), and 5 µl of the first PCR product under the same amplification conditions as the first PCR but with a modification of the extension time (90 seconds at 72°C) per cycle. Secondary PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA, http://www1.qiagen.com) and were directly sequenced by using the BigDye Terminator v3.1 Cycle Sequencing Kit on a 3100 DNA sequencer (Applied Biosystems) according to the manufacturer's manual. We used the same primers described in our recent report [30] for sequencing.

Mutation Scoring and Donor or Recipient CD34+ Clone Recognition
Sequences were aligned by the DNAstar program (DNASTAR Inc., Madison, WI, http://www.dnastar.com) and were proofread by eye. Mutations were scored relative to the revised Cambridge Reference Sequence [33]. Sites showing a heteroplasmy (coexisting of wild-type and mutant) of at least 10% mutant according to the sequencing chromatographs were scored. The length mutations of C tract in region 16184–16193 due to 16189T>C mutation were not scored in single cells. The same data quality control that we described previously [30] was applied in this study. We used the network method [34], which has been widely applied in population genetics to demonstrate intraspecific mtDNA variation, to represent the relationship among haplotypes identified in single CD34+ cells. Each haplotype is represented by a circle, with area proportional to the frequency of haplotype; different circles (haplotypes) are connected by lines proportional to the mutation distance. The resulting network facilitates visualization of different CD34+ cells from donor and recipient. The length mutations of C tract in region 303–309 in the second hypervariable segment of mtDNA control region was discarded when constructing the network. The other sporadic insertions and deletions (indels) and length mutations of the dinucleotide AC repeat in region 515–524 were included in the network analysis. Because the aggregate or consensus sequences of sibling sample pairs are identical (supplemental online Table 1), CD34+ clones could only be identified as deriving from recipient or donor by recipient- or donor-specific somatic mutations. In several cases, nucleotide substitutions were observed in CD34+ cells from both donor and recipient before transplant and were regarded as either germline mutations or recurrent parallel mutations. mtDNA sequence heterogeneity level in each sample was measured by the number of haplotypes per 100 single CD34+ cells. This index reflects the total number of mutations that have occurred within a cell population and not the frequency of the cell clone harboring certain mutation(s) [29, 30].


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Maintenance of CD34+ Heterogeneity After Transplantation
Immediately apparent from a cursory examination of the mtDNA variation profiles in all patients (Table 1) was a high level of heterogeneity in CD34+ cells from recipients after transplantation. mtDNA sequence heterogeneity of CD34+ cells in the four patients studied soon after transplantation persisted during the period of rapid reconstitution and proliferation, perhaps reflective of continued activity of host marrow and the input donor CD34+ cells. The two patients who were studied at late time points after transplantation showed that heterogeneity had not been lost. This pattern made observations of individual clones feasible and suggests that either sufficient donor HSCs are engrafted (together with the remaining recipient HSCs) to be representative of the observed heterogeneity or (considerably less likely) a similar pattern of heterogeneity could rapidly occur despite transplantation of limited numbers of CD34+ cells.

Tracing Donor- and Recipient-Specific CD34+ Cell Clones
A site-by-site audit of the mtDNA mutation information in single CD34+ cells from each of the six sample pairs (which includes samples from both the donor and recipient before and after the transplant; supplemental online Tables 1 and 2), as represented by the network profile in Figure 1Go and supplemental online Figure 1, offers a convenient method to track clones characterized by donor- or recipient-specific somatic mutations.


Figure 1
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  		Figure 1. Network profile of mitochondrial DNA (mtDNA) haplotypes (CD34+ cell clones) observed in single CD34+ cell populations from the donor and recipient before and after the nonmyeloidablative allogeneic stem cell transplantation. (A): BURRC-BURDC, aplastic anemia; (B): QUIDC-MAUWC, gastric cancer; (C): CABO-CABL, renal cell cancer. The networks were drawn according to the median-joining method [34] based on the mutation information in supplemental online Table 2. The length mutations of C tract in region 303–309 in the mtDNA control region was not considered. The order of mutations on the branch is arbitrary. Each circle represents an mtDNA haplotype or a CD34+ cell clone as recognized by mtDNA mutation(s) in a population of CD34+ cells. The area of the circle is proportional to the frequency of the haplotype and is further specified by the number of cells sharing that haplotype. For instance, "4#" means this haplotype was found in four single cells in that sample. In each network, the haplotype in the center is the major type that has the consensus or aggregate sequence. We used the following colors to mark the source of the clones and mutations: red, clones/mutations in donor and recipient after transplantation; dark blue, clones/mutations in recipient before and after transplantation; light blue, clones/mutations in donor and recipient before transplantation; light pink, clones/mutations in the same donor collected at different time points; and yellow (refer to supplemental online Fig. 1B for sample ANDGC), clones/mutations in the same recipient after transplantation collected at different time points. Abbreviations: del, deletion; ins, insertion.

 


Figure 1A
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  		Figure 1. (Continued).

 

Patients Studied Soon After Transplantation.   In the patient with aplastic anemia (BURDC), the genetic profile of CD34+ cells after transplantation for nearly 8 months strongly resembled that of the donor, harboring a similar frequency of donor clones with specific somatic mutations 56, 195, 251, and 292; none of these clones was found in CD34+ cells from the recipient before transplantation. Persistence of the recipient's CD34+ cell clones after transplantation was less recognizable, although the relatively high frequency of cells with a heteroplasmic dinucleotide AC insertion at 523–524 possibly reflected a contribution from the recipient, or alternatively was due to clonal expansion of a genetically similar clone present in both the donor and recipient (Fig. 1GoA).

In samples from the sex-mismatched gastric cancer patient/donor pair (QUIDC-WAUWC), mutations 16093, 16053, 16304, and 16189 and length mutations of a C tract in region 568–573 (not including the C-tract change in region 303–309) were detected in CD34+ cells from both the donor and recipient before transplantation, likely representing inherited germline mutations or parallel somatic events. Recipient-specific clones, which were recognizable by somatic mutations 237 and 16519, remained detectable at stable levels in CD34+ cells for 5 months after transplantation (Fig. 1GoB).

In samples from the renal cell cancer patient/donor pair (GREHC-GREAC), fewer mutations were observed in single CD34+ cells compared with the other five sibling pairs who were older. Mutations 16293 and 16537, presumably donor in origin, could be detected in the recipient 3 months after transplantation (supplemental online Fig. 1A).

Cells were collected from samples obtained from the pancreatic cancer patient/donor pair (HERJC-ANDGC) at 1 and 6 months after transplantation; CD34+ clones from the donor (recognized by variants 310 and 16183) and the recipient (variants 179 and 16408) were identified after transplantation in the recipient, although the majority of the observed mutations were not sufficiently informative to permit distinguishing a donor or recipient source (supplemental online Fig. 1B).

Patients Studied Late After Transplantation.   To determine whether CD34+ cell clones marked by certain mutations could be sustained for a long period of time, we analyzed mtDNA mutations in CD34+ cells from two renal cell cancer patients who survived for more than 3 and 8 years, respectively, after NASCT and donor samples collected at corresponding time points. Of interest, continued chimerism of donor and recipient clones, as recognized by donor-specific variants 131 and 469 and recipient-specific variants 16238 and 16288, was detected in the recipient 8 years after transplantation, which is especially remarkable given that T-cell and myeloid chimerism rapidly converted from mixed to full donor in origin shortly following infusion of the donor allograft in this patient, as assessed by PCR of polymorphic minisatellite regions [32]. Although age-dependent mutation accumulation seemingly occurred in the donor during this 8-year period (Fig. 1GoC; Table 1), clones in the donor bearing the sentinel mutations at sites 227, 309, 541, 16221, and 16272 remained stable and neither expanded nor disappeared (Fig. 1GoC).

In the recipient with renal cancer who has survived for more than 3 years, CD34+ chimerism could not be assessed, as we failed to distinguish donor- or recipient-specific clones in any post-transplant sample. Although two clones characterized by mutations 16043 and 16222 were present in both the donor and recipient after transplantation, these variants were also present in the recipient before the procedure. The two CD34+ clones in the donor containing mutations 137 and 16093 were repeatedly observed during the 3-year interval (supplemental online Fig. 1C).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Mitochondrial DNA has been widely used as a marker in molecular anthropology and forensic science [35–37]. Normal cells, tissues, and tumors acquire mtDNA somatic mutations over time [28, 30, 31, 38–41], and many laboratories have attempted to correlate such acquired mutations with disease or specific organ dysfunction [38, 40]. In the present study, we have attempted to use mtDNA control region somatic sequence variants in single CD34+ cells to mark and trace HSC reconstitution after NASCT. Because donors and recipients were HLA-matched siblings and their mtDNA sequences were in aggregate identical (supplemental online Table 1), the donor and recipient could be distinguished only by individual-specific somatic mutations. By comparing the mutations observed in CD34+ clones from the donor and the recipient before and after transplantation, the source of clones could in principle be identified according to their unique mtDNA somatic mutations (or haplotypes). Preferential clonal expansion of certain CD34+ cells bearing specific nucleotide substitutions in the recipient would potentially be recognizable based on the change in frequency of the cells harboring the mutation, as we have observed for single leukemia blasts [30]. Compared with other methods for assessing hematopoietic chimerism after transplantation [22, 25, 42–49], the single-cell analysis method allows discrimination among different HSC clones, although its sensitivity is dependent on the abundance of mtDNA mutations within the CD34+ cell population and their presence in primitive HSCs or in committed progenitors.

A critical concern regarding the explanation for observed somatic mutations in single CD34+ cells is whether genetic variants remain stable in the clones during mtDNA duplication and cell self-renewal, or the observed mutations are artifacts introduced by PCR and/or contamination. Furthermore, distinguishing a parallel mtDNA mutation from a marker mutation in different cells is difficult. Using donor and recipient of different matrilines would help to reduce these uncertainties. Three lines of evidence suggest the relative stability of marker mutations in CD34+ cells. First, donor CD34+ clones might have a similar potential for maintenance after transplantation in the recipient, as we observed that donor-specific CD34+ clones characterized by mutations 56, 195, 251, and 292 had all repopulated an aplastic anemia patient by 8 months after transplantation (Fig. 1GoA). Second, in the two donors sampled twice over 3- and 8-year intervals, although the majority of mutations in single CD34+ cells differed, several mutations occurring with a similar frequency were observed at both time points (Fig. 1GoC; supplemental online Fig. 1C), presumably of the same clonal origin. Third, a comparison of mtDNA variation in CD34+ cells, T cells, B cells, and granulocytes from the same healthy donors showed that some of the mutations in differentiated cells were present in CD34+ cells [28]. Therefore, the persistence of specific CD34+ clones originating from either the donor or recipient after transplantation could be quantitatively inferred by examination of specific mtDNA somatic mutations, but no simple statistical test could be developed to quantify the observed pattern.

Consistent with the use of nonmyeloablative, reduced-intensity conditioning [16, 17], mixed chimerism of CD34+ cells in recipient was detected after transplantation based on mtDNA mutation patterns (Fig. 1Go; supplemental online Fig. 1). The recipient with aplastic anemia acquired a high prevalence of donor CD34+ cell chimerism shortly after transplantation. In contrast, the imprint of CD34+ cells of donor origin was less recognizable in the patients with gastric cancer (MAUWC), pancreatic cancer (ANDGC), and renal cell cancer (GREAC and THOMC), all of whom had relatively normal marrow function that was affected only by prior chemotherapy and not by disease or surgical treatment. It is important to note that we examined only a limited number of CD34+ cells and that there was a paucity of mutations specific to the donor/recipient with which to track CD34+ cell origin; these limitations potentially may have biased our observations. In addition, the CD34+ population is heterogeneous and contains both primitive HSCs and committed progenitors; only primitive HSCs contribute to long-term multilineage hematopoietic reconstitution [3, 50], and mutations in these cells might be more consistently detected over time. The observation of donor and recipient CD34+ cells (recognized by specific mutations) in patient CABL 8 years after transplantation was unexpected, as full donor chimerism in T cells and myeloid cells was documented on multiple occasions in post-transplant blood samples by PCR-based chimerism of minisatellite regions [32]. Differences in chimerism of more differentiated lymphohematopoietic cells compared with CD34+ cells in the recipient after transplantation could potentially be the consequence of variability in susceptibility of different cell populations to donor immune-mediated graft-versus-host hematopoietic effect [23, 51]. The persistence of mixed chimerism of donor and recipient hematopoiesis for many years has been reported in patients who received transplants to cure their thalassemia [46] and acute lymphoblastic leukemia [52].

Discarding potential disease and therapeutic effect on CD34+ cells, a comparison of the mtDNA mutation levels in cells from sibling samples might provide information as to whether the nuclear genetic background affected the rate and prevalence of mtDNA somatic mutation in single HSCs during aging, similar to the pattern that was recently observed in our murine study [29]. An approximate comparison of the mutation levels (number of haplotypes defined by nucleotide substitutions in 100 CD34+ cells) showed no correlation between mtDNA mutation number and age in 16 healthy adults (ages 25 to 65 years old; reported in this study and [28, 30]), and adult samples could seemingly be sorted into two groups, with mutation levels ranging from 6.5 to 12.8 and from 29.7 to 42.6, respectively. These results could be explained by modulation from the nuclear genetic background [29]. Indeed, the sibling sample pair GREHC-GREAC had a similar but lower level of mutation within a population of CD34+ cells compared with the other five sibling pairs. An aging effect on the mtDNA mutation level in single CD34+ cells from the same adult donor may require a very prolonged period of time, as we observed an increase of mtDNA mutation during the 8-year period in donor CABO but not during the 3-year interval observation for another donor (KIRGC). Also intriguing is that a small portion of mutations in single CD34+ cells was shared between sibling samples and seemed to be exclusive to that family (Fig. 1Go; supplemental online Fig. 1). The exact reason for the occurrence of these mutations in HSCs remains to be resolved.

In summary, recipients maintained a high level of overall CD34+ cell heterogeneity after NASCT. Direct comparison of CD34+ clones in the donor and recipient before and after transplantation based on their specific mtDNA somatic mutations has confirmed the expected early mixed chimerism associated with nonmyeloablative transplantation in HSCs, which persisted for a prolonged period in one patient. The observation that recipients acquire a similar profile and frequency of donor-specific mtDNA mutations in CD34+ cells following transplantation suggests that each transplanted donor HSC clone might have an equal potential for trafficking, homing, and survival in the host. These observations provide insights into hematopoietic stem cell activity after NASCT.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Leigh Samsel for technical assistance and Rose Goodwin for help in collecting the patient samples. We are also grateful to Dr. Colin Wu for statistical assistance.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Dick JE, Magli MC, Huszar D et al. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell 1985;42:71–79.[CrossRef][Medline]

  2. Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986;45:917–927.[CrossRef][Medline]

  3. Dick JE, Lapidot T. Biology of normal and acute myeloid leukemia stem cells. Int J Hematol 2005;82:389–396.[CrossRef][Medline]

  4. Lapidot T, Dar A, Kollet O. How do stem cells find their way home? Blood 2005;106:1901–1910.[Abstract/Free Full Text]

  5. Papayannopoulou T. Current mechanistic scenarios in hematopoietic stem/progenitor cell mobilization. Blood 2004;103:1580–1585.[Abstract/Free Full Text]

  6. Camargo FD, Chambers SM, Drew E et al. Hematopoietic stem cells do not engraft with absolute efficiencies. Blood 2006;107:501–507.[Abstract/Free Full Text]

  7. Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.[CrossRef][Medline]

  8. Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science 1996;273:242–245.[Abstract]

  9. Spangrude GJ, Brooks DM, Tumas DB. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: In vivo expansion of stem cell phenotype but not function. Blood 1995;85:1006–1016.[Abstract/Free Full Text]

  10. Abkowitz JL, Catlin SN, McCallie MT et al. Evidence that the number of hematopoietic stem cells per animal is conserved in mammals. Blood 2002;100:2665–2667.[Abstract/Free Full Text]

  11. Gordon MY, Lewis JL, Marley SB. Of mice and men ... and elephants. Blood 2002;100:4679–4680.[Free Full Text]

  12. Shepherd BE, Guttorp P, Lansdorp PM et al. Estimating human hematopoietic stem cell kinetics using granulocyte telomere lengths. Exp Hematol 2004;32:1040–1050.[CrossRef][Medline]

  13. Abkowitz JL, Persik MT, Shelton GH et al. Behavior of hematopoietic stem cells in a large animal. Proc Natl Acad Sci U S A 1995;92:2031–2035.[Abstract/Free Full Text]

  14. Prchal JT, Prchal JF, Belickova M et al. Clonal stability of blood cell lineages indicated by X-chromosomal transcriptional polymorphism. J Exp Med 1996;183:561–567.[Abstract/Free Full Text]

  15. Morris E, Thomson K, Craddock C et al. Outcomes after alemtuzumab-containing reduced-intensity allogeneic transplantation regimen for relapsed and refractory non-Hodgkin lymphoma. Blood 2004;104:3865–3871.[Abstract/Free Full Text]

  16. Childs R, Chernoff A, Contentin N et al. Regression of metastatic renal-cell carcinoma after nonmyeloablative allogeneic peripheral-blood stem-cell transplantation. N Engl J Med 2000;343:750–758.[Abstract/Free Full Text]

  17. Childs R, Clave E, Contentin N et al. Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: Full donor T-cell chimerism precedes alloimmune responses. Blood 1999;94:3234–3241.[Abstract/Free Full Text]

  18. Peggs KS, Hunter A, Chopra R et al. Clinical evidence of a graft-versus-Hodgkin's-lymphoma effect after reduced-intensity allogeneic transplantation. Lancet 2005;365:1934–1941.[CrossRef][Medline]

  19. Giralt S, Thall PF, Khouri I et al. Melphalan and purine analog-containing preparative regimens: Reduced-intensity conditioning for patients with hematologic malignancies undergoing allogeneic progenitor cell transplantation. Blood 2001;97:631–637.[Abstract/Free Full Text]

  20. Slavin S, Nagler A, Naparstek E et al. Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 1998;91:756–763.[Abstract/Free Full Text]

  21. Deeg HJ, Maris MB, Scott BL et al. Optimization of allogeneic transplant conditioning: Not the time for dogma. Leukemia 2006;20:1701–1705.[CrossRef][Medline]

  22. Baron F, Sandmaier BM. Chimerism and outcomes after allogeneic hematopoietic cell transplantation following nonmyeloablative conditioning. Leukemia 2006;20:1690–1700.[CrossRef][Medline]

  23. Griffith LM, McCoy JP Jr, Bolan CD et al. Persistence of recipient plasma cells and anti-donor isohaemagglutinins in patients with delayed donor erythropoiesis after major ABO incompatible non-myeloablative haematopoietic cell transplantation. Br J Haematol 2005;128:668–675.[CrossRef][Medline]

  24. Carvallo C, Geller N, Kurlander R et al. Prior chemotherapy and allograft CD34+ dose impact donor engraftment following nonmyeloablative allogeneic stem cell transplantation in patients with solid tumors. Blood 2004;103:1560–1563.[Abstract/Free Full Text]

  25. Mackinnon S, Barnett L, Bourhis JH et al. Myeloid and lymphoid chimerism after T-cell-depleted bone marrow transplantation: Evaluation of conditioning regimens using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA. Blood 1992;80:3235–3241.[Abstract/Free Full Text]

  26. Mackinnon S, Barnett L, Heller G et al. Minimal residual disease is more common in patients who have mixed T-cell chimerism after bone marrow transplantation for chronic myelogenous leukemia. Blood 1994;83:3409–3416.[Abstract/Free Full Text]

  27. Shin MG, Kajigaya S, Tarnowka M et al. Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes. Blood 2004;103:4466–4477.[Abstract/Free Full Text]

  28. Ogasawara Y, Nakayama K, Tarnowka M et al. Mitochondrial DNA spectra of single human. CD34+ cells, T cells, B cells, and granulocytes. Blood 2005;106:3271–3284.[Abstract/Free Full Text]

  29. Yao Y-G, Ellison FM, McCoy JP et al. Age-dependent accumulation of mtDNA mutations in murine hematopoietic stem cells is modulated by the nuclear genetic background. Hum Mol Genet 2007;16:286–294.[Abstract/Free Full Text]

  30. Yao Y-G, Ogasawara Y, Kajigaya S et al. Mitochondrial DNA sequence variation in single cells from leukemia patients. Blood 2007;109:756–762.[Abstract/Free Full Text]

  31. Shin MG, Kajigaya S, McCoy JP Jr et al. Marked mitochondrial DNA sequence heterogeneity in single CD34+ cell clones from normal adult bone marrow. Blood 2004;103:553–561.[Abstract/Free Full Text]

  32. Childs RW, Clave E, Tisdale J et al. Successful treatment of metastatic renal cell carcinoma with a nonmyeloablative allogeneic peripheral-blood progenitor-cell transplant: Evidence for a graft-versus-tumor effect. J Clin Oncol 1999;17:2044–2049.[Abstract/Free Full Text]

  33. Andrews RM, Kubacka I, Chinnery PF et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 1999;23:147.[CrossRef][Medline]

  34. Bandelt H-J, Forster P, Röhl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 1999;16:37–48.[Abstract]

  35. Macaulay V, Hill C, Achilli A et al. Single, rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes. Science 2005;308:1034–1036.[Abstract/Free Full Text]

  36. Yao Y-G, Kong Q-P, Bandelt H-J et al. Phylogeographic differentiation of mitochondrial DNA in Han Chinese. Am J Hum Genet 2002;70:635–651.[CrossRef][Medline]

  37. Salas A, Bandelt H-J, Macaulay V et al. Phylogeographic investigations: The role of trees in forensic genetics. Forensic Sci Int 2007;168:1–13.[CrossRef][Medline]

  38. Wallace DC. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine. Annu Rev Genet 2005;39:359–407.[CrossRef][Medline]

  39. Chinnery PF, Samuels DC, Elson J et al. Accumulation of mitochondrial DNA mutations in ageing, cancer, and mitochondrial disease: Is there a common mechanism? Lancet 2002;360:1323–1325.[CrossRef][Medline]

  40. Taylor RW, Turnbull DM. Mitochondrial DNA mutations in human disease. Nat Rev Genet 2005;6:389–402.[Medline]

  41. Chomyn A, Attardi G. MtDNA mutations in aging and apoptosis. Biochem Biophys Res Commun 2003;304:519–529.[CrossRef][Medline]

  42. Blazar BR, Orr HT, Arthur DC et al. Restriction fragment length polymorphisms as markers of engraftment in allogeneic marrow transplantation. Blood 1985;66:1436–1444.[Abstract/Free Full Text]

  43. Durnam DM, Anders KR, Fisher L et al. Analysis of the origin of marrow cells in bone marrow transplant recipients using a Y-chromosome-specific in situ hybridization assay. Blood 1989;74:2220–2226.[Abstract/Free Full Text]

  44. Suttorp M, Schmitz N, Dreger P et al. Monitoring of chimerism after allogeneic bone marrow transplantation with unmanipulated marrow by use of DNA polymorphisms. Leukemia 1993;7:679–687.[Medline]

  45. Price CM, Colman SM, Kanfer EJ. Persistence of multilineage host haemopoiesis following allogeneic bone marrow transplantation. Br J Haematol 1995;90:465–468.[Medline]

  46. Andreani M, Manna M, Lucarelli G et al. Persistence of mixed chimerism in patients transplanted for the treatment of thalassemia. Blood 1996;87:3494–3499.[Abstract/Free Full Text]

  47. Alizadeh M, Bernard M, Danic B et al. Quantitative assessment of hematopoietic chimerism after bone marrow transplantation by real-time quantitative polymerase chain reaction. Blood 2002;99:4618–4625.[Abstract/Free Full Text]

  48. Hochberg EP, Miklos DB, Neuberg D et al. A novel rapid single nucleotide polymorphism (SNP)-based method for assessment of hematopoietic chimerism after allogeneic stem cell transplantation. Blood 2003;101:363–369.[Abstract/Free Full Text]

  49. Fredriksson M, Barbany G, Liljedahl U et al. Assessing hematopoietic chimerism after allogeneic stem cell transplantation by multiplexed SNP genotyping using microarrays and quantitative analysis of SNP alleles. Leukemia 2004;18:255–266.[CrossRef][Medline]

  50. Wang JC, Dick JE. Cancer stem cells: Lessons from leukemia. Trends Cell Biol 2005;15:494–501.[CrossRef][Medline]

  51. Clave E, Rocha V, Talvensaari K et al. Prognostic value of pretransplantation host thymic function in HLA-identical sibling hematopoietic stem cell transplantation. Blood 2005;105:2608–2613.[Abstract/Free Full Text]

  52. Singer JW, Keating A, Ramberg R et al. Long-term stable hematopoietic chimerism following marrow transplantation for acute lymphoblastic leukemia: A case report with in vitro marrow culture studies. Blood 1983;62:869–872.[Abstract/Free Full Text]





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