Intragraft Chimerism Following Composite Tissue Allograft

Journal of Surgical Research 157, 129-135 (2009) doi:10.1016/j.jss.2008.06.026

RESEARCH REVIEW Intragraft Chimerism Following Composite Tissue Allograft Keiichi Muramatsu, M.D.,1 Ryutaro Kuriyama, M.D., and Toshihiko Taguchi, M.D. Department of Orthopedic Surgery, Yamaguchi University School of Medicine, Yamaguchi, Japan Submitted for publication April 21, 2008

Until now, more than 35 hand transplants have been performed in humans and have generated much public interest. Cell traffic from the recipient into the graft, so-called intragraft chimerism, appears to play a major role in graft acceptance and graft rejection. Little is known about cell migration following extremity allografts. In this review, recent experimental studies are presented for intragraft chimerism of the extremity allograft. Technical tools for detecting recipient cells in the graft were: (1) immunohistochemistry, (2) karyotyping, (3) fluorescent in situ hybridization, (4) polymerase chain reaction, and (5) transgenic animals. This study demonstrates that recipient-derived cells gradually repopulate into grafted skin, bone tissues, bone marrow, and endothelial cells, but muscle, periosteum, and cartilage tissues retain donor cell origin. © 2009 Elsevier Inc. All rights reserved. Key Words: chimerism; extremity allograft; polymerase chain reaction; transgenic animals. INTRODUCTION

Graft acceptance occurs when a transplanted organ gradually becomes less immunogenic. Several hypotheses have been proposed to explain this process. In 1962, Medawar [1] first hypothesized that graft acceptance might be the result of replacement of the endothelial cells of the graft by those of the recipient. Replacement of donor cells by recipient-derived cells in solid organ allografts has been reported in several papers [2–7]. It is now speculated that circulating stem cells derived from the bone marrow of recipients are able to differentiate into graft cells [8, 9]. However, the 1

To whom correspondence and reprint requests should be addressed at Department of Orthopedic Surgery, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. E-mail: [email protected].

clinical implications of graft cell replacement and the factors affecting the timing and extent of intragraft chimerism remain poorly understood. In this paper, we review intragraft chimerism following composite tissue allograft and discuss how the immune response can be modulated by changes in antigenicity of the limb allograft from pure donor to partial recipient. TECHNICAL TOOLS FOR DETECTING RECIPIENT-DERIVED CELLS IN THE GRAFT

The availability of a technical tool to demonstrate the presence of chimerism is essential for studies of intragraft chimerism. Commonly used techniques are: (1) immunohistochemistry [5], (2) karyotyping [10], (3) fluorescent in situ hybridization (FISH) [7], (4) polymerase chain reaction (PCR) [11], and (5) transgenic animals [12]. Sedmak et al. [3] have also studied recipient endothelialization of clinical renal allografts using immunohistochemical staining for ABO-blood group antigens. However, this technique is limited to ABO nonidentical transplants and cannot be used in animal studies. Porter [13] used karyotyping in liver transplants, but this technique requires the presence of dividing cells in metaphase. At present, FISH for the Y-chromosome is probably the most common technique in sex-mismatched transplantation to distinguish donor and recipient cells on a histological slide. Hruban et al. [2] applied FISH technique for the Y-chromosome in a clinical study of sex-mismatched cardiac transplantation. Lagaaij et al. [4] studied clinical kidney allografts using three different techniques: immunohistochemistry for MHC class-I antigen, immunohistochemistry for ABO-blood-group antigens, and FISH for X and Y chromosomes.

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However, FISH in calcified tissue is technically demanding and probes specific for rat Y-chromosome are not commercially available [14, 15]. Although the efficiency of hybridization of the Y-chromosome probe in intact cells may reach 99%, it is not as high in tissue sections, with some nuclei being destroyed during sectioning. PCR is the easiest and most reliable tool to prove the existence of different genetic material in the same body [16 –18]. We previously attempted competitive PCR study using Sry and glyceraldehyde phosphate dehydrogenase (GAPDH) specific primers to determine the ratio of male to female cells in bone allografts [11, 12]. The control PCR study demonstrated very high sensitivity for the Y-chromosome, detecting male DNA at a ratio of ⱖ1 in 1000. A visible GAPDH band demonstrates a successful PCR reaction. Comparison of the Sry and GAPDH bands allows accurate, semiquantitative evaluation of male-to-female cell ratios suitable for quantitative analysis of intragraft chimerism [19, 20]. Recently, numerous investigations of cell migration following transplantation have been performed using transgenic (Tg) mice that express reporter genes such as green fluorescent protein (GFP) [21, 22] or ␤-galactosidase (LacZ) [23, 24]. Although transgenic mice technology is well established, these animals are too small for experimental hind limb transplantation. Rats are more suitable due to their larger body size. Hakamata et al. [21] were the first to develop transgenic GFP and LacZ rats. The GFP rat is transfected with a specific marker gene derived from jellyfish that does not require chemical substrate for visualization [25]. GFP gene expression is easily and elegantly detected in these animals using fluorescence microscopy and 489 nm excitation light. The presence and ratio of donor to recipient cells can be also evaluated by semiquantitative PCR technique. Ajiki et al. [26, 27] have previously used GFP transgenic rats for marking donor cells and have used FACScan to study graft cell migration to the recipient following whole-limb allografting. They demonstrated migration of donor-derived hematopoietic stem cells from rat limb allografts to the recipient bone marrow. GFP gene expression, however, varied in each tissue and organ. Visceral organs such as liver, spleen, and kidney showed little or no GFP expression, whereas marked GFP intensity was observed in the pancreatic-, cardiac-, skeletal muscle, and skin tissues [21, 22]. The distribution of GFP expression provides valuable information on cell migration to and from whole-limb allografts. We initially used the LacZ-Tg rat to investigate intragraft chimerism in whole-limb allografts [9, 28, 29]. LacZ-positive cells are readily detected histologically by ␤-galactosidase (X-gal) staining [23, 24]. Their presence and the ratio of donor to recipient cells can also be evaluated by semiquantitative PCR technique. The

background of LacZ-Tg rats is inbred DA and hence there are no immunological barriers with other inbred DA rats. Although LacZ-Tg rats have distinct advantages for cell migration studies, some problems remain. First, LacZ gene expression varies in different tissues and organs. According to a studies by Takahashi et al. [23] and Inoue et al. [24], visceral organs such as lung, liver, spleen and kidney, brain, small intestine, vessels, spinal cord, bone marrow cells and leukocytes showed little or no LacZ expression, whereas marked expression was observed in the pancreatic, cardiac, skeletal muscle, cartilage and skin tissues. The second problem is the apparent absence of LacZ gene expression in osseous tissue, a problem that is also common to the GFP-Tg rat. Although we decalcified the mineral component of bone with EDTA, LacZ expression was not detected in bone cells. It is unclear whether the decalcification process disturbed gene expression or whether bone cells do not express the LacZ gene. At present, PCR technique may be the only method capable of detecting the LacZ gene in hard tissues. INTRAGRAFT CHIMERISM IN SOLID ORGAN TRANSPLANT AND EXTREMITY ALLOGRAFT

There are two possible directions for cell traffic following transplantation: donor to recipient and recipient to donor [30, 31]. Until now, numerous papers have focused on cell movement from donor to recipient, the so-called systemic chimerism [32–35]. Detection of intragraft chimerism following organ transplantation and differentiation of recipient cells in the graft has been attempted by several authors. Kashiwagi et al. [10] used a karyotyping technique in human liver allografts, demonstrating that hepatocytes and major vessel endothelium retained their donor specificity, whereas the macrophage system was rapidly replaced with recipient cells. Using in situ hybridization techniques for the Y-chromosome, Hruban et al. [2] demonstrated that cardiac myocytes and a majority of endothelial cells retain donor specificity. Theise et al. [36] showed that extrahepatic stem cells, probably bone marrow-derived, contributed to the proliferation of hepatocytes. Intragraft chimerism is likely to be incomplete in solid organ transplantations. Multiple studies have shown that populations of stem cells or progenitor cells exist in the adult and are able to develop into multiple, adult-type tissues during growth and regeneration [37–39]. Ten years have passed since the first hand allotransplant was performed in humans, and more than 35 allogeneic hands have since been transplanted [40–44]. These raise new problems that are specific to handtransplant cases. Among them, a major concern is that patients feel their new hand as alien hands. Whereas visceral organ allografts are invisible, hands are cos-

MURAMATSU ET AL.: INTRAGRAFT CHIMERISM FOLLOWING COMPOSITE TISSUE ALLOGRAFT

FIG. 1. Whole hind-limb allograft model from DA rat to Lewis. (Color version of figure is available online.)

metically important body parts. To resolve this issue, we must in the future define whether the donor’s stranger hand can become the recipient’s own hand. This phenomenon constitutes intragraft chimerism. Little is known about the lineage of living cells in grafts and the regulation of cell turnover following hand transplant. Replacement by cells from the circulation or from adjoining normal tissue is possible. Undifferentiated multipotential cells may reach the graft by direct migration from adjacent tissues or through anastomosed arteries. Cellular differentiation or proliferation of these multipotent cells within the graft is then possible. Composite allografts consist of various tissue combinations, including muscle, nerve, tendon, skin, bone, cartilage, and bone marrow. Cell turnover and renewal may be different in each tissue and in various damaged conditions following the transplant procedure. To date, there have been few scientific studies that describe cell lineages in limb grafts. BONE CHIMERISM

Bone tissue undergoes constant cell remodeling throughout life. Osteoprogenitor cells originate from a mesenchymal stem-cell line and reside mainly in the bone periosteum and marrow [45– 47]. Cell traffic from the recipient into the grafted bone therefore seems natural if one considers this mechanism of bone remodeling. However, little is known on the subject of bone chimerism. We attempted to define the cell lineage of the grafted bone in the rat vascularized tibial bone and whole hind-limb allograft model (Fig. 1) [8, 11]. Cell repopulation was assessed by semiquantitative PCR using Sry primers in sex-mismatched transplantation. This study demonstrated that the original bone cells are gradually replaced to a large extent with recipient-

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derived cells (Table 1). Interestingly, this repopulation begins simultaneously in all portions of the bone graft. This even distribution suggests the cells are supplied via the nutrient artery rather than from adjacent marrow or periosteum. The replacement process occurs to a surprising extent, such that by 24 wk, approximately 99% of the original bone cell line was replaced with circulation-derived cells. Similar results were obtained in the rat hind limb allograft model. Cell replacement of grafted bone in the rat is therefore a rapid process. Osteonal remodeling is likely to be slower in humans [45]. The life span of an osteoblast varies from a few d to 3 mo, depending on the timing of incorporation into the new bone as an osteocyte. The life span of an osteocyte or a lining cell in cortical bone varies from several years to several decades. To our knowledge, there have been no reports that clearly describe the life span of rat bone cells. Rat osteocytes in cortical bone are likely to survive at least 6 mo to 1 y. Our observations on graft cell replacement at 24 wk suggest rapid cell turnover in vascularized bone grafts. Osteotomy is likely to stimulate the migration, proliferation, and differentiation of undifferentiated cells into osteoblasts that are responsible for healing of bone. In future work, clear histological demonstration will be necessary to prove the cell replacement process of the grafted bone allograft. ENDOTHELIAL CHIMERISM

Several studies have addressed the question of endothelial cell replacement in solid organ transplantation [4 –7]. The consensus was that the endothelium remained mostly of donor origin. Clouston et al. [48] reported that endothelial cell chimerism was not observed up to 410 d following liver transplantation. However, other studies found some evidence of endothelial cell chimerism in biopsies obtained within one year after liver transplant [5]. Koestner et al. [49] and O’Connell et al. [50] demonstrated that some heart transplant cases showed endothelial replacement by recipient cells at varying times and frequencies. Tanaka et al. [5] showed a time-dependent increase in the number of recipient-derived endothelial cells in the capillaries of the portal vein after liver transplant. We attempted to demonstrate endothelial chimerism in TABLE 1 Cell Lineages of Limb Allograft Graft tissues in which donor origin cells remain permanently

Graft tissues in which recipient cells gradually migrate into

Muscle Cartilage Periosteum

Bone Skin Bone marrow Endothelial cell

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into new endothelial cells [51]. These endothelial progenitor cells may contribute to the renewal of damaged endothelial cells. MUSCLE CHIMERISM

FIG. 2. In non-GFP limb graft, the endothelial cell layer of capillaries revealed new GFP expression at 18 mo after transplant [12]. (Color version of figure is available online.)

the extremity allograft using GFP transgenic rats [12]. Fortunately, GFP expression is quite high in the endothelium, and we observed GFP expression in the endothelial cells of GFP-negative graft transplanted into GFP-positive recipient, thus demonstrating endothelial chimerism (Fig. 2). In a study of kidney transplantation, Sedmak et al. [3] reported an association between endothelial damage induced by rejection and endothelial chimerism. The vascular endothelium is always damaged by ischemic-reperfusion injury. Hristov et al. [51] reported in a recent review that endothelial cell repair from ischemia may occur via the migration and proliferation of surrounding mature endothelial cells. However, mature endothelial cells are terminally differentiated and therefore have a low potential for proliferation and substitution of damaged cells. Recent studies indicate that peripheral blood contains bone marrow-derived progenitor cells that have the potential to differentiate

The skeletal muscle comprises self-renewing tissues. In the normal state, growth and repair are mediated by a resident population of self-maintaining myogenic tissue stem cells, the so-called satellite cells [52]. This renewal mechanism is different from that of bone tissue renewal. Satellite cells are situated between the basal lamina and plasma membrane, and are locally activated by myofiber degeneration induced by ischemia or operative intervention. From our experiments, the grafted muscle of donor limbs was likely to have been severely damaged by warm ischemia and reperfusion injury because limb grafts were swollen for 2 wk after transplant [9, 12]. Under the influence of growth factors released by inflammatory cells and macrophages, satellite cells proliferate and differentiate into myoblasts, which then further differentiate and fuse to form myotubes and subsequently myofibers, thus promoting muscle regeneration. However, the self-renewing potential of satellite cells is limited [52, 53]. More recent studies on skeletal muscle regeneration suggest that myogenic cells can be derived from stem cells in ectopic bone marrow, although their participation in healing is likely to be minor [54]. Our study using the LacZ-Tg rat model clearly showed the muscle component remained of donor origin up to at least 1 y after transplant (Fig. 3) [9]. However, another study using the GFP-Tg rat model showed that partial muscle fibers expressed cells of recipient origin and were located around small vessels [12]. New stem cells may be supplied through these vessels. SKIN CHIMERISM

Although little is known about the kinetics of renewal of cutaneous tissue under steady-state condi-

FIG. 3. In transplanted GFP (A) and LacZ (B) limbs, the muscle fiber showed retained marked expression of GFP and LacZ at 18 mo after transplant [9, 12]. (Color version of figure is available online.)

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FIG. 4. In non-GFP limb graft, new GFP expression was noted especially at hair follicles 18 mos after transplant [12]. (Color version of figure is available online.)

tions, skin is generally considered to be self-renewing [55, 56]. Review of the recent literature suggests that growth and repair are mediated by upper regions of the outer root sheath of hair follicles, or the so-called bulge region [55]. These cells divide at a slow rate to sustain both self-renewal and growth of differentiated tissue. Our studies using Tg rats and PCR clearly showed that donor skin cells were gradually replaced by recipient cells [9, 12]. Interestingly, these cells centered on the hair follicle (Fig. 4). A new line of cell supply to the skin stem cell is likely to arise from an ectopic mesenchymal stem cell. Recent studies show that bone marrowderived cells are found in the epidermis and hair follicle, but these do not appear to undergo clonal expansion unless the tissue is injured. Merad et al. [56] used a chimeric mouse model and transplanted bone marrow cells into irradiated mice and investigated Langerhans cell renewal in the skin. Their results suggest that donor-derived Langerhans cells can proliferate and regenerate under steady-state conditions. However, under conditions that severely deplete epidermal Langerhans cells, these cells were rapidly replaced by circulating recipient cells from the peripheral blood. The graft is likely to suffer damage through surgical intervention and ischemia during anastomosis of the femoral vessels. These injuries might induce renewal via the recruitment of ectopic cells. In the clinic, Kanitakis et al. [57] performed the first human hand allograft and investigated the repopulation of Langerhans cell in the epidermis of the allograft. Cells of recipient origin within the allograft were detected by immunolabeling with an antibody specific for the recipient’s HLA antigen. Their results showed that the Langerhans cells present in the skin of the allograft remained of donor origin over a 4- to 5-y period, suggesting a slow rate of repopulation of human epidermal Langerhans cells from the recipient. The speed of cell repopulation of cutaneous tissue is likely to be species-dependent. Longer follow-up of pa-

tients may reveal that some or all of the epidermal population in the allograft is replaced by recipient cells. Skin has proven to be the most antigenic component in the limb allograft [58]. In our GFP-Tg study [12], the grafted limb was not rejected by recipients up to 18 mo following transplant, even after withdrawal of FK506 therapy for 6 mo. We speculate that the immune response is modulated by changes in antigenicity of the limb allograft from pure donor to partial recipient, thus potentially accounting for the lack of rejection reactions. EXTREMITY CHIMERISM IN OTHER COMPONENTS

The cell turnover and remodeling of articular chondrocytes has been well known. Chondrocytes are derived from mesenchymal cells that differentiate during

FIG. 5. In LacZ limb allograft, all chondrocytes stained with X-gal retained marked blue expression of LacZ Femur condyle at 1 y post-transplant [9]. (Color version of figure is available online.)

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skeletal morphogenesis and develop to form chondrocytes [59]. Because adult cartilage lacks blood vessels, there is no migration of undifferentiated cells from the blood. In addition to lacking blood vessels, cartilage lacks undifferentiated cells within the tissue. The only cell type found in articular cartilage is the highly differentiated chondrocyte, which has limited capacity for proliferation and migration because it is encased within the dense collagen-proteoglycan extracellular matrix of the tissue. Our data using the LacZ-Tg rat showed that chondrocytes remain of donor origin at least 1 y after transplant (Fig. 5). This observation is not surprising considering the cell morphogenesis of chondrocytes. As demonstrated by several earlier papers [8, 12, 60], we found that recipient-derived bone marrow cells migrated into the bone marrow of the limb graft. The repopulation of marrow cells seemed unexpectedly fast and was maintained more than 1 y after transplant. Cell turnover in the periosteum was largely unknown until recently. Our data using GFP-Tg rats demonstrates that the periosteum of the limb graft retains its donor cell origin even at 18 mo post-transplant, suggestive of self renewing tissue [12]. REFERENCES

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