Donor bone-marrow CXCR4+ Foxp3+ T-regulatory cells are essential for costimulation blockade-induced long-term survival of murine limb transplants.

Vascularized composite allotransplantation (VCA) allows tissue replacement after devastating loss but is currently limited in application and may be more widely performed if maintenance immunosuppression was not essential for graft acceptance. We tested whether peri-transplant costimulation blockade could prolong VCA survival and required donor bone-marrow cells, given that bone-marrow might promote graft immunogenicity or graft-versus-host disease. Peritransplant CD154 mAb/rapamycin (RPM) induced long-term orthotopic hindlimb VCA survival (BALB/c->C57BL/6), as did CTLA4Ig/RPM. Surprisingly, success of either protocol required a bone-marrow-associated, radiation-sensitive cell population, since long-bone removal or pre-transplant donor irradiation prevented long-term engraftment. Rejection also occurred if Rag1-/- donors were used, or if donors were treated with a CXCR4 inhibitor to mobilize donor BM cells pre-transplant. Donor bone-marrow contained a large population of Foxp3+ T-regulatory (Treg) cells, and donor Foxp3+ Treg depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed diphtheria toxin receptor, restored rejection with either protocol. Rejection also occurred if CXCR4 was deleted from donor Tregs pre-transplant. Hence, long-term VCA survival is possible across a full MHC disparity using peritransplant costimulation blockade-based approaches, but unexpectedly, the efficacy of costimulation blockade requires the presence of a radiation-sensitive, CXCR4+ Foxp3+ Treg population resident within donor BM.

Cryopreservation and Transplantation of Vascularized Composite Transplants: Unique Challenges and Opportunities.

Vascularized composite allotransplantation is the ultimate reconstructive tool when no other means of reconstruction are available. Despite its immense potential, the applicability of vascularized composite allotransplantation is hampered by high rejection rates and the requirement for high doses of immunosuppressive drugs that are associated with severe adverse effects and death. Because this is a non-life-saving procedure, widespread use of vascularized composite allotransplantation demands methods that will allow the reduction or elimination of immunosuppressive therapy. Efficient methods for the cryopreservation of biological cells and tissues have been sought for decades. The primary challenge in the preservation of viable tissue in a frozen state is the formation of intracellular and extracellular ice crystals during both freezing and thawing, which cause irreversible damage to the tissue. Recent proof-of-concept transplantations of a complete cryopreserved and thawed hindlimb in a rat model have demonstrated the potential of such methods. In the current review, the authors discuss how limb cryopreservation can attenuate or eliminate allograft rejection by either enabling better human leukocyte antigen matching or by adaptation of clinical tolerance protocols such as mixed chimerism induction. Also, the authors discuss the possible advantages of cryopreservation in autologous tissue salvage and cryopreservation following trauma. Clinical-grade cryopreservation may revolutionize the field of reconstruction, organ banking, and complex traumatic limb injury management.

Effect of different sustained bone morphogenetic protein-2 release kinetics on bone formation in poly(propylene fumarate) scaffolds.

To investigate the effect of sustained bone morphogenetic protein-2 (BMP-2) release kinetics on bone formation in poly(propylene fumarate) (PPF) scaffolds, different poly(lactic-co-glycolic acid) (PLGA) microspheres were used as delivery vehicles. All PPF scaffolds had the same 75% porous structure, while the degradation rate of the embedded PLGA microspheres was changed to tailor BMP-2 release by varying the lactic-to-glycolic acid (L:G) ratio in the copolymer. Four PLGA microsphere formulations with 50/50, 65/35, 75/25, and 85/15 L:G ratios and varying in vivo degradation rates were fabricated. The in vitro and in vivo BMP-2 release kinetics were determined by analyzing radiolabeled 125 I-BMP-2. Biological activity of released BMP-2 was tested using a W20-17 cell culture model in vitro and a subcutaneous rat model in vivo. Corresponding outcome parameters were defined as capacity to increase the in vitro AP activity in weekly consecutive cell cultures over 14 weeks and the in vivo bone formation after 7 and 14 weeks. The PLGA/PPF composites showed similar biological activity and BMP-2 release profiles in vitro. In vivo, PPF/PLGA 85:15 composite released significantly less BMP-2 per time point in the first weeks. Micro-CT and histological analysis after 7 and 14 weeks of implantation showed bone formation, which significantly increased over time for all composites. No significant differences were seen between the composites. Overall, the results of this study show that small differences in BMP-2 sustained release had no significant effect on BMP-2 osteogenic efficacy in PPF/PLGA composites. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 477-487, 2018.

Free-Tissue Transfer for the Reconstruction of War-Related Extremity Injuries: A Systematic Review of Current Practice.

OBJECTIVES: Extremity injuries in combat zones have devastating sequelae. The increasing survival of war-zone casualties, combined with rapid advances in microsurgery, means that there is a growing role for free-tissue reconstruction. We systematically reviewed the current practices in microsurgical reconstruction of combat-related extremity injuries, focusing on free-flap types, timing of surgery, and outcomes. METHODS: We conducted a PubMed search of the terms "War" and "Reconstruction," identifying 21st century studies on subacute/delayed free-flap repair, to reflect the idiosyncrasies of modern warfare. Case reports and studies exclusively describing craniofacial and thoracoabdominal injuries were excluded. RESULTS: A total of 11 studies fulfilled our inclusion criteria. In 9 studies, patients were repatriated/transferred to specialist facilities for treatment; in 2 studies, reconstruction was performed within combat/austere environments. The number of free-flaps described per study ranged from 6 to 233 (Total = 501). Latissimus dorsi flaps were most commonly used (43.7%). The average time to definitive reconstruction ranged from 9.6 days to 3 years, being delayed to address life-threatening injuries. The average free-flap success rate was 95.5% (range = 88.9%-100%). CONCLUSION: Combat-associated extremity injuries are characterized by extensive tissue loss and gross contamination. Despite this, microsurgical reconstruction results in minimal morbidity and successful outcomes. Large, multicenter studies are necessary to corroborate these findings and establish definitive management guidelines.

Lessons learned from the first quadruple extremity transplantation in the world.

BACKGROUND: Limb transplantation is emerging as a promising area of surgery and is an indispensable alternative for prosthetic rehabilitation of amputees, the severity of which is increasing because of combat-related injuries. Successful unilateral and bilateral limb transplantations have already been performed before this operation. METHODS: We performed the first ever quadruple limb transplantation in February 2012. The limbs procured from a 40-year-old man heart-beating donor were transplanted to a 27-year-old male patient who was a quadruple amputee for the last 14 years because of an electrical injury. RESULTS: To shorten the ischemic period to a minimum, 3 separate microsurgery teams worked simultaneously. All extremities were reperfused within 8 hours of procurement, and the operation lasted for 12 hours. Metabolic load was managed by hemodialysis. One hour after the completion of the operation, cardiac arrest developed, resuscitation of which necessitated median sternotomy and temporary partial cardiopulmonary support. Despite the removal of the transplanted limbs and all efforts including continuous hemodialysis, plasmapheresis, and extracorporeal membrane oxygenation, the patient died on the fourth day after transplantation in a clinical condition of severe systemic inflammation. CONCLUSIONS: The problems we faced were difficulty of vascular access for invasive monitoring and fluid replacement, and the severe systemic inflammation effects of which could not be dealt with, despite aggressive supportive treatment. We hope that our experience will enlighten the surgeons who are willing to extend the limits of limb transplantation and serve the success of future operations.

Histomorphometric evaluation of ischemia-reperfusion injury and the effect of preservation solutions histidine-tryptophan-ketoglutarate and University of Wisconsin in limb transplantation.

BACKGROUND: The effect of cold ischemia (CI) in vascularized composite allotransplantation is unknown. We herein assess tissue-specific damage, acceptable CI time, and the effect of preservation solutions in a syngenic rat hindlimb transplant model. METHODS: Lewis rat limbs were flushed and stored for 2, 10, or 30 hr CI in saline, histidine-tryptophan-ketoglutarate or University of Wisconsin preservation solution before transplantation. Morphologic alterations, inflammation, and damage of the individual tissues were analyzed on day 10 using histomorphology, confocal, light, and transmission-electron microscopy. RESULTS: Two-hour CI led to mild inflammation of tissues on day 10, whereas 10-hr and 30-hr CI resulted in massive inflammation and tissue damage. Although muscle was mainly affected after prolonged CI (≥10 hr), nerve was affected in all CI groups. A perineural cell infiltrate, hypercellular appearance, pronounced vacuolization, and mucoid degeneration, appearing as Wallerian degeneration, were observed. Staining with propidium iodide and Syto 16 revealed a decrease in viable muscle cell nuclei in the anterior tibial muscle on day 10 in all groups, which was most pronounced in 10-hr and 30-hr CI animals. Transmission-electron microscopy indicated that a large number of mitochondria were degenerated in the 10-hr and 30-hr CI groups. Histidine-tryptophan-ketoglutarate preservation solution slightly decreased inflammation and tissue damage compared to University of Wisconsin-treated and saline-treated animals, especially in skin and muscle when CI times did not exceed 10 hr. CONCLUSION: Severe inflammation and tissue damage are observed after prolonged CI in muscle and nerve. Ischemia times in vascularized composite allotransplantation should be kept as short as possible and certainly below 10 hr.

Insights from computational modeling in inflammation and acute rejection in limb transplantation.

Acute skin rejection in vascularized composite allotransplantation (VCA) is the major obstacle for wider adoption in clinical practice. This study utilized computational modeling to identify biomarkers for diagnosis and targets for treatment of skin rejection. Protein levels of 14 inflammatory mediators in skin and muscle biopsies from syngeneic grafts [n = 10], allogeneic transplants without immunosuppression [n = 10] and allografts treated with tacrolimus [n = 10] were assessed by multiplexed analysis technology. Hierarchical Clustering Analysis, Principal Component Analysis, Random Forest Classification and Multinomial Logistic Regression models were used to segregate experimental groups. Based on Random Forest Classification, Multinomial Logistic Regression and Hierarchical Clustering Analysis models, IL-4, TNF-α and IL-12p70 were the best predictors of skin rejection and identified rejection well in advance of histopathological alterations. TNF-α and IL-12p70 were the best predictors of muscle rejection and also preceded histopathological alterations. Principal Component Analysis identified IL-1α, IL-18, IL-1ß, and IL-4 as principal drivers of transplant rejection. Thus, inflammatory patterns associated with rejection are specific for the individual tissue and may be superior for early detection and targeted treatment of rejection.

Vascularized bone graft for oncological reconstruction of the extremities: review of the biological advantages.

Vascularized bone graft (VBG) is a form of vascularized bone marrow transplant in which the bone marrow is surgically grafted with its microenvironment intact. Due to the preservation of cellular viability, VBG have significant advantages over non-vascularized bone grafts. Free vascularized fibula grafts have superior material properties and tolerate infection. Bone healing can be accomplished in a shorter period, even in an irradiated bed. In addition to these properties, VBG has other biological advantages that are not always familiar to oncological surgeons. Hypertrophic change can be divided into reactive and adaptive hypertrophy. Early hypertrophy is associated with donor-derived cells, whereas later remodeling is associated with recipient-derived cells. VBG has significant advantages in enhancing neo-revascularization of necrotic bone. We reviewed VBG from a novel viewpoint that stems from our basic research.

Phase I trial: the use of autologous cultured adipose-derived stroma/stem cells to treat patients with non-revascularizable critical limb ischemia.

BACKGROUND AIMS: Non-revascularizable critical limb ischemia (CLI) is the most severe stage of peripheral arterial disease, with no therapeutic option. Extensive preclinical studies have demonstrated that adipose-derived stroma cell (ASC) transplantation strongly improves revascularization and tissue perfusion in ischemic limbs. This study, named ACellDREAM, is the first phase I trial to evaluate the feasibility and safety of intramuscular injections of autologous ASC in non-revascularizable CLI patients. METHODS: Seven patients were consecutively enrolled, on the basis of the following criteria: (i) lower-limb rest pain or ulcer; (ii) ankle systolic oxygen pressure <50 or 70 mm Hg for non-diabetic and diabetic patients, respectively, or first-toe systolic oxygen pressure <30 mm Hg or 50 mm Hg for non-diabetic and diabetic patients, respectively; (iii) not suitable for revascularization. ASCs from abdominal fat were grown for 2 weeks and were then characterized. RESULTS: More than 200 million cells were obtained, with almost total homogeneity and no karyotype abnormality. The expressions of stemness markers Oct4 and Nanog were very low, whereas expression of telomerase was undetectable in human ASCs compared with human embryonic stem cells. ASCs (10(8)) were then intramuscularly injected into the ischemic leg of patients, with no complication, as judged by an independent committee. Trans-cutaneous oxygen pressure tended to increase in most patients. Ulcer evolution and wound healing showed improvement. CONCLUSIONS: These data demonstrate the feasibility and safety of autologous ASC transplantation in patients with objectively proven CLI not suitable for revascularization. The improved wound healing also supports a putative functional efficiency.

Nerve regeneration in rat limb allografts: evaluation of acute rejection rescue.

BACKGROUND: Successful nerve regeneration is critical to the functional success of composite tissue allografts. The present study was designed to characterize the effect of acute rejection on nerve regeneration and functional recovery in the setting of orthotopic limb transplantation. METHODS: A rat orthotopic limb transplantation model was used to evaluate the effects of acute rejection on nerve regeneration and motor recovery. Continuous administration of FK506 (full suppression), administration of FK506 for the first 8 of 12 weeks (late rejection), or delayed administration of FK506/dexamethasone following noticeable rejection (early rejection) was used to preclude or induce rejection following limb transplantation. Twelve weeks postoperatively, nerve regeneration was assessed by means of histomorphometric analysis of explanted sciatic nerve, and motor recovery was assessed by means of evoked muscle force measurement in extensor digitorum longus muscle. RESULTS: A single episode of acute rejection that occurs immediately or late after reconstruction does not significantly alter the number of regenerating axonal fibers. Acute rejection occurring late after reconstruction adversely affects extensor digitorum longus muscle function in composite tissue allografts. CONCLUSIONS: Collected data reinforce that adequate immunosuppressant administration in cases of allogeneic limb transplantation ensures levels of nerve regeneration and motor functional recovery equivalent to that of syngeneic transplants. Prompt rescue following acute rejection was further demonstrated not to significantly affect nerve regeneration and functional recovery postoperatively. However, instances of acute rejection that occur late after reconstruction affect graft function. In total, the present study begins to characterize the effect of immunosuppression regimens on nerve regeneration and motor recovery in the setting of composite tissue allografts.

Imparting regenerative capacity to limbs by progenitor cell transplantation.

The frog Xenopus can normally regenerate its limbs at early developmental stages but loses the ability during metamorphosis. This behavior provides a potential gain-of-function model for measures that can enhance limb regeneration. Here, we show that frog limbs can be caused to form multidigit regenerates after receiving transplants of larval limb progenitor cells. It is necessary to activate Wnt/ß-catenin signaling in the cells and to add Sonic hedgehog, FGF10, and thymosin ß4. These factors promote survival and growth of the grafted cells and also provide pattern information. The eventual regenerates are not composed solely of donor tissue; the host cells also make a substantial contribution despite their lack of regeneration competence. Cells from adult frog legs or from regenerating tadpole tails do not promote limb regeneration, demonstrating the necessity for limb progenitor cells. These findings have obvious implications for the development of a technology to promote limb regeneration in mammals.

Proangiogenic features of Wharton's jelly-derived mesenchymal stromal/stem cells and their ability to form functional vessels.

Mesenchymal stromal/stem cells derived from human Wharton's jelly (WJ-MSC) have emerged as a favorable source for autologous and allogenic cell therapy. Here, we characterized the proangiogenic features of WJ-MSCs and examined their ability to form functional vessels in in vivo models. First, we examined whether WJ-MSCs express endothelial and smooth muscle cell specific markers after culture in endothelial growth media. WJ-MSCs expressed an endothelial specific marker, VEGFR1, at mRNA and protein levels, but did not express other specific markers (VEGFR2, Tie2, vWF, CD31, and VE-cadherin). Rather, WJ-MSCs expressed smooth muscle cell specific markers, α-SMA, PDGFR-ß and calponin, and were unable to form tube-like structures with lumen on Matrigel. WJ-MSCs secreted growth factors including angiogenin, IGFBP-3, MCP-1, and IL-8, which stimulated endothelial proliferation, migration, and tube formation. When WJ-MSCs suspended in Matrigel were implanted into nude mice, it led to formation of functional vessels containing erythrocytes after 7 days. However, implantation of endothelial cell-suspended Matrigel resulted in no perfused vessels. The implanted WJ-MSCs were stained positively for calponin or PDGFR-ß and were located adjacent to the lining of mouse endothelial cells that were stained with labeled BS-lectin B4. In a murine hindlimb ischemia model, the transplantation of MSCs (5×10(5)cells) into the ischemic limbs improved perfusion recovery and neovascularization of the limbs compared to control group. Therefore, the results suggest that WJ-MSCs promote neovascularization and perfusion by secreting paracrine factors and by functioning as perivascular precursor cells, and that WJ-MSCs can be used efficiently for cell therapy of ischemic disease.

Small animal models of xenotransplantation.

Organ transplantation has become a successful and acceptable treatment for end-stage organ failure. Such success has allowed transplant patients to resume a normal lifestyle. The demands for transplantation have been steadily increasing, as more patients and new diseases are being deemed eligible for treatment via transplantation. However, it is clear that human organs will never meet the increasing demand of transplantation. Therefore, scientists must continue to pursue alternative therapies and explore new treatments to meet the growing demand for the limited number of organs available. Transplanting organs from animals into humans (xenotransplantation) is one such therapy. The observed enthusiasm for xenotransplantation, irrespective of the severe shortage of human organs and tissues available for transplantation, can be said to stem from at least two factors. First, there is the possibility that animal organs and tissues might be less susceptible than those of humans to the recurrence of disease processes. Second, a xenograft might be used as a vehicle for introducing novel genes or biochemical processes which could be of therapeutic value for the transplant recipient.To date, millions of lives have been saved by organ transplantation. These remarkable achievements would have been impossible without experimental transplantation research in animal models. Presently, more than 95% of organ transplantation research projects are carried out using rodents, such as rats and mice. The key factor to ensure the success of these experiments lies in state-of-the art experimental surgery. Small animal models offer unique advantages for the mechanistic study of xenotransplantation rejection. Currently, multiple models have been developed for investigating the different stages of immunological barriers in xenotransplantation. In this chapter, we describe six valuable small animal models that have been used in xenotransplantation research. The methodology for the small animal model establishment includes animal selection, preoperative care, anesthesia, postoperative care, and detailed procedures.

Biologics and donor bone marrow cells for targeted immunomodulation in vascularized composite allotransplantation: a translational trial in swine.

INTRODUCTION: Bone marrow (BM) infusion following organ transplantation is a prerequisite for potential donor-antigen-specific tolerance induction. We developed a preclinical swine model to determine the optimal dose of BM cells to achieve microchimerism. Furthermore, induction therapy was optimized by augmenting the BM infusion with biologics in the form of costimulatory blockade: cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig). MATERIALS AND METHODS: Yucatan miniature swine (n = 12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30, or 60 million cells per kilogram of whole unmodified BM. The optimal dose of BM cell infusion (BMT) was then applied to subsequent experiments evaluating the addition of CTLA4lg. Group 1 (control) received no treatment. Group 2 received FK506 only; group 3 received irradiation, BMT, and FK506; group 4 received FK506 and CTLA4-lg. RESULTS: Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 million cells per kilogram (P = .0001). Transplanted animals in group 1 rejected the allograft 5 to 8 days after transplantation. Group 2 rejected the allograft (skin and muscle) 30 to 32 days after transplantation (2 days after cessation of immunosuppression). Group 3 rejected the skin portion of the allograft at 50, 52, and 53 days posttransplant. Remaining allograft components (muscle, bone, nerve, vessel) survived indefinitely. Group 4 animals demonstrated significantly prolonged muscle survival beyond 150 days posttransplant; the skin component survived past 150 days in two of three animals. Skin and muscle histology in all long-term surviving animals were normal. CONCLUSIONS: BM cell infusion with 60 million cells per kilogram results in stable levels of microchimerism. The addition of costimulatory blockade (CTLA4lg) prolonged allograft skin survival and overall graft survival. Such targeted immunomodulatory protocols might facilitate immune tolerance and eliminate the need for multidrug immunosuppression to maintain graft survival after vascularized composite allotransplantation.

Secondary ectopic transfer for replantation salvage after severe wound infection.

BACKGROUND: Ectopic transfer has been described as a salvage procedure in failing replants. The experience in three cases of infected failing replantations treated with secondary temporary ectopic transfer of the replanted part is presented. METHODS: Three patients with replanted traumatic amputations (one transhumeral, one transmetacarpal, and one transtibial) that developed severe wound infections and thrombosis of the anastomoses were treated with urgent ectopic transfer of the replanted part. The ectopic recipient vessels were the femoral, posterior tibial, and the descending branch of the lateral femoral circumflex arteries. The stumps were surgically cleansed and the ectopically replanted parts were retransferred some days later. RESULTS: The infection reccurred in one case and the replant (transmetacarpal) was lost. The two other cases were successfully retransferred orthotopically, 9 and 20 days later, respectively. In one case (transtibial) multiple additional surgical procedures were necessary. Functional results in these two cases were acceptable. CONCLUSIONS: Delayed ectopic transfer is a useful, yet demanding technique for the salvage of complicated replants in the context of severe wound infection and vascular thrombosis or impending failure. Given the complexity of the procedure it should only be considered in selected cases.

Advances in the development of experimental composite tissue transplantation models.

The preclinical experimental models of composite tissue allograft (CTA) have rapidly developed in the past years. When microsurgical techniques were established, researchers focused on immunomodulatory protocols that overcome the immunologic barrier between the allogenic donor and recipient. To test immunologic response, functional recovery, and technical feasibility, experimental CTA has been performed in different models, including rodents, large animals, and nonhuman primates. In the experimental studies, researchers are focused on tolerance-inducing strategies based on immunosuppressive protocols allowing for widespread application in the clinic. In this review, authors analyzed the current knowledge of immunologic aspects and tolerance-inducing strategies in CTA experimental models, including single components such as skin or vascularized bone allograft versus CTA containing multiple tissues such as experimental limb and face transplants, and emphasized their relevance and applicability to the clinical scenario.

[Organ transplantation, composite tissue allotransplantation, and plastic surgery]. / Organtransplantation, Gewebetransplantation und plastische Chirurgie.

Transplantations play an increasing role for plastic reconstructive surgeons. The increasing number of solid organ transplantations and the improved long-term survival rates lead to increased numbers of these patients also undergoing plastic and reconstructive procedures. Free flap transfer in solid organ transplant patients is feasible with no higher risk to both transplant function and postoperative complications than for nontransplant patients, even during immune suppression. Composite tissue allotransplantation (CTA) is an evolving field in plastic reconstructive surgery with hands, arms, partial faces, abdominal walls, and knee joints being transferred in clinical settings. However only an interdisciplinary approach using all available resources in highly selected patients after exhausting all other plastic reconstructive procedures is able to achieve reasonable results. The potential complications of long-term immune suppression and patient compliance have to be balanced with the expected and achieved functional result of CTA, whose procedures must be discussed as a potential tissue or organ transplantation, given the legal and logistic implications. The interdisciplinary cooperation of transplant surgeons, microsurgeons, psychologists, and ergo- and physiotherapists is mandatory to achieve successful CTA results.

Body wall morphogenesis: limb-genesis interferes with body wall-genesis via its influence on the abaxial somite derivatives.

The vertebrate body wall is regionalized into thoracic and lumbosacral/abdominal regions that differ in their morphology and developmental origin. The thoracic body wall has ribs and intercostal muscles, which develops from thoracic somites, whereas the abdominal wall has abdominal muscles, which develops from lumbosacral somites without ribs cage. To examine whether limb-genesis interferes with body wall-genesis, and to test the possibility that limb generation leads to the regional differentiation, an ectopic limb was induced in the thoracic region by transplanting prospective limb somatopleural mesoderm of Japanese quail between the ectoderm and somatopleural mesoderm of the chick prospective thoracic region. This ectopic limb generation induced the somitic cells to migrate into the ectopic limb mesenchyme to become its muscles and caused the loss of distal thoracic body wall (sterno-distal rib and distal intercostal muscle), without causing any significant effect on the more proximal region (proximal rib, vertebro-distal rib and proximal intercostal muscle). According to a new primaxial-abaxial classification, the proximal region is classified as primaxial and the distal region, as well as limb, is classified as abaxial. We demonstrated that ectopic limb development interfered with body wall development via its influence on the abaxial somite derivatives. The present study supports the idea that the somitic cells give rise to the primaxial derivatives keeping their own identity and fate, whereas they produce the abaxial derivatives responding to the lateral plate mesoderm.