Implications of an Altered Gut Microbiome in Rat Experimental Vascularized Composite Transplantation.

OBJECTIVES: Vascularized composite allotransplantation is a reconstructive option after severe injury but is fraught with complications, including transplant rejection due to major histocompatibility complex mismatch in the context of allogeneic transplant, which in turn is due to altered immuno-inflammation secondary to transplant. The immunosuppressant tacrolimus can prevent rejection. Because tacrolimus is metabolized predominantly by the gut, this immunosuppressant alters the gut microbiome in multiple ways, thereby possibly affecting immunoinflammation. MATERIALS AND METHODS: We performed either allogeneic or syngeneic transplant with or without tacrolimus in rats. We quantified protein-level inflammatory mediators in the skin, muscle, and plasma and assessed the diversity of the gut microbiome through 16S RNA analysis at several timepoints over 31 days posttransplant. RESULTS: Statistical analysis highlighted a complex interaction between major histocompatibility complex and tacrolimus therapy on the relative diversity of the microbiome. Time-interval principal component analysis indicated numerous significant differences in the tissue characteristics of inflammation and gut microbiome that varied over time and across experimental conditions. Classification and regression tree analysis suggested that both inflammatory mediators in specific tissues and changes in the gut microbiome are useful in characterizing the temporal dynamics of posttransplant inflammation. Dynamic network analysis highlighted unique changes in Methanosphaera that were correlated with Peptococcusin allogeneic transplants with and without tacrolimus versus Prevotella in syngeneic transplant with tacrolimus, suggesting that alterations in Methanosphaera might be a biomarker of vascularized composite allotransplant rejection. CONCLUSIONS: Our results suggest a complex interaction among major histocompatibility complex, local and systemic immuno-inflammation, and tacrolimus therapy and highlight the potential for novel insights into vascularized composite allotransplant from computational approaches.

OPTN/SRTR 2022 Annual Data Report: Vascularized Composite Allograft.

This year's chapter on vascularized composite allograft (VCA) encompasses reviews of data collected from 2014 (when VCA was included in the Final Rule) through 2022. The present Annual Data Report shows that the number of VCA recipients in the United States continues to be small and has remained consistent from the prior report. The data continue to be limited by sample size, with trends persistently demonstrating a predominance of White males in the young/middle-aged population as both donors and recipients for nonuterus VCA transplants, and White women younger than 35 years as the predominant recipients of uterus transplant. Similar to the 2021 report, there were only eight failed uterus grafts and one failed nonuterus VCA graft reported from 2014 through 2022. Standardization of definitions of success and failure as well as outcome measures for the different VCA types remain unmet needs in VCA transplantation.

Lymphadenopathy and lymph node rejection following facial vascularized composite allotransplantation.

BACKGROUND: Apart from the skin, little is known about the immunological processes in deeper tissues, which are typically not accessible to biopsy and inspection, of vascularized composite allografts (VCAs). Face transplant patients develop prominent adenopathy shortly after transplantation that resolves over time. The mechanisms underlying this process are not understood. MATERIALS AND METHODS: A retrospective cohort study was conducted on 9 patients who underwent 10 facial VCAs at the Brigham and Women's Hospital, Boston, MA, between April 2009 and July 2019. Clinical, radiological, and histological data related to lymphadenopathy of the head and neck were reviewed. RESULTS: Patients who received donor-derived lymph nodes (LNs) developed bilateral lymphadenopathy of the submental or submandibular superficial LNs. Median time of presentation was POD18 (range POD6-POM3). Notably, bilateral adenopathy of the neck was not observed in later stages of follow-up (mean follow-up, 115 months). Histology of 3 LNs showed increased histiocytes and apoptosis, with the features reminiscent of necrotizing histiocytic lymphadenitis, and B and T lymphocytes (mostly CD8 + T) admixed with CD163 + histiocytes and dendritic cells. Molecular chimerism analysis in one case showed the coexistence of donor (81%) and recipient (19%) derived lymphocytes. Granzyme B (GZMB) expression confirmed the presence of increased cytotoxic T cells in this LN sample. CONCLUSION: Our data suggested the involvement of an immunological process within the donor-derived LNs after facial allotransplantation between the recipient and donor cells. GZMB expression suggested LN rejection that can occurred independently of skin rejection. This finding supports the need to better define the role of donor-derived immune cells in the context of allograft rejection.

Developing online communication training to request donation for vascularized composite allotransplantation (VCA): improving performance to match new US organ donation targets.

BACKGROUND: Approaching families of dying or newly deceased patients to donate organs requires specialized knowledge and a mastery of relational communication. As the transplantation field has progressed, Donation Professionals (DPs) are also leading conversations with family decision makers (FDMs) about the donation of uncommon anatomical gifts, such as face, hands, genitalia, referred to as Vascularized Composite Allotransplants (VCA) without much training or experience. To address the need for training, we adapted and beta tested an evidenced-based communication training program for donation discussions to VCA requests. The overarching goal of Communicating Effectively about Donation for Vascularized Composite Allotransplantation (CEaD-VCA) is to increase the number of VCA authorizations and to improve the socioemotional outcomes of FDMs. METHODS: We developed CEaD-VCA, an online, on-demand training program based on the previously tested, evidenced-based communication skills training program designed to train DPs to have conversations about solid organ donation. The training was modified utilizing data from a national telephone survey with DPs and results of 6 focus groups conducted with members of the general public. The survey and focus groups assessed knowledge, attitudes, and barriers to VCA donation. The training was shaped by a partnership with a leading industry partner, the Gift of Life Institute.™ RESULTS: Using the results as a guide, the existing CEaD training program, consisting of interactive eLearning modules, was adapted to include technical information about VCA, foundational communication skills, and two interactive example VCA donation request scenarios to facilitate active learning. Forty-two DPs from two partner Organ Procurement Organizations (OPOs) participated in the beta test of CEaD-VCA. Pre- and post-test surveys assessed the impact of the training. CONCLUSIONS: The training was scored highly by DPs in effectiveness and ease of use. This project created a standardized, accessible, and comprehensive training for DPs to communicate about VCA donation. CEaD-VCA is an example of how to develop a communication skills training for difficult conversations utilizing input from stakeholders, guided by communication theory. It also demonstrates how gaps in communication skills during medical education can be filled utilizing advanced online Learning Management Systems. The training specifically addresses new CMS rules concerning OPO performance metrics.

First-in-Human Whole-Eye Transplantation: Ensuring an Ethical Approach to Surgical Innovation.

As innovations in the field of vascular composite allotransplantation (VCA) progress, whole-eye transplantation (WET) is poised to transition from non-human mammalian models to living human recipients. Present treatment options for vision loss are generally considered suboptimal, and attendant concerns ranging from aesthetics and prosthesis maintenance to social stigma may be mitigated by WET. Potential benefits to WET recipients may also include partial vision restoration, psychosocial benefits related to identity and social integration, improvements in physical comfort and function, and reduced surgical risk associated with a biologic eye compared to a prosthesis. Perioperative and postoperative risks of WET are expected to be comparable to those of facial transplantation (FT), and may be similarly mitigated by immunosuppressive protocols, adequate psychosocial support, and a thorough selection process for both the recipient and donor. To minimize the risks associated with immunosuppressive medications, the first attempts in human recipients will likely be performed in conjunction with a FT. If first-in-human attempts at combined FT-WET prove successful and the biologic eye survives, this opens the door for further advancement in the field of vision restoration by means of a viable surgical option. This analysis integrates recent innovations in WET research with the existing discourse on the ethics of surgical innovation and offers preliminary guidance to VCA programs considering undertaking WET in human recipients.

The Nuances of Hand Transplantation After Sepsis.

Vascularized composite allotransplantation (VCA) of the upper extremity is an established restorative procedure for selected patients with acquired upper limb loss. The majority of upper limb VCAs performed worldwide have been for victims of various forms of trauma. However, in the developed world, amputation following severe sepsis seems to be an increasingly common indication for referral to hand transplant programs. Unlike trauma patients with isolated limb injuries, patients with amputations as a complication of sepsis have survived through a state of global tissue hypoperfusion and multisystem organ failure with severe, enduring effects on the entire body's physiology. This article reviews the unique considerations for VCA candidacy in postsepsis patients with upper limb amputation. These insights may also be relevant to postsepsis patients undergoing other forms of transplantation or to VCA patients requiring additional future solid organ transplants.

Local Delivery of Adipose Stem Cells Promotes Allograft Survival in a Rat Hind-Limb Model of Vascularized Composite Allotransplantation.

BACKGROUND: Adipose stem cells (ASCs) are a promising cell-based immunotherapy because of their minimally invasive harvest, high yield, and immunomodulatory capacity. In this study, the authors investigated the effects of local versus systemic ASC delivery on vascularized composite allotransplant survival and alloimmune regulation. METHODS: Lewis rats received hind-limb transplants from Brown Norway rats and were administered donor-derived ASCs (passage 3 or 4, 1 × 10 6 cells/rat) locally in the allograft, or contralateral limb, or systemically at postoperative day 1. Recipients were treated intraperitoneally with rabbit anti-rat lymphocyte serum on postoperative days 1 and 4 and daily tacrolimus for 21 days. Limb allografts were monitored for clinical signs of rejection. Donor cell chimerism, immune cell differentiation, and cytokine expression in recipient lymphoid organs were measured by flow cytometric analysis. The immunomodulation function of ASCs was tested by mixed lymphocyte reaction assay and ASC stimulation studies. RESULTS: Local-ASC-treated recipients achieved significant prolonged allograft survival (85.7% survived >130 days; n = 6) compared with systemic-ASC and contralateral-ASC groups. Secondary donor skin allografts transplanted to the local-ASC long-term surviving recipients accepted permanently without additional immunosuppression. The increases in donor cell chimerism and regulatory T-cells were evident in blood and draining lymph nodes of the local-ASC group. Moreover, mixed lymphocyte reaction showed that ASCs inhibited donor-specific T-cell proliferation independent of direct ASC-T-cell contact. ASCs up-regulated antiinflammatory molecules in response to cytokine stimulation in vitro. CONCLUSION: Local delivery of ASCs promoted long-term survival and modulated alloimmune responses in a full major histocompatibility complex-mismatched vascularized composite allotransplantation model and was more effective than systemic administration. CLINICAL RELEVANCE STATEMENT: ASCs are a readily available and abundant source of therapeutic cells that could decrease the amount of systemic immunosuppression required to maintain limb and face allografts.

Vascularized Composite Allograft Versus Prosthetic for Reconstruction After Facial and Hand Trauma: Comparing Cost, Complications, and Long-term Outcome.

ABSTRACT: In the past decade, vascularized composite allotransplantation (VCA) has become clinical reality for reconstruction after face and hand trauma. It offers patients the unique opportunity to regain form and function in a way that had only been achieved with traditional reconstruction or with the use of prostheses. On the other hand, prostheses for facial and hand reconstruction have continued to evolve over the years and, in many cases, represent the primary option for patients after hand and face trauma. We compared the cost, associated complications, and long-term outcomes of VCA with prostheses for reconstruction of the face and hand/upper extremity. Ultimately, VCA and prostheses represent 2 different reconstructive options with distinct benefit profiles and associated limitations and should ideally not be perceived as competing choices. Our work adds a valuable component to the general framework guiding the decision to offer VCA or prostheses for reconstruction after face and upper extremity trauma.

Mimicking Clinical Rejection Patterns in a Rat Osteomyocutaneous Flap Model of Vascularized Composite Allotransplantation.

INTRODUCTION: Graft loss in vascularized composite allotransplantation (VCA) is more often associated with vasculopathy and chronic rejection (CR) than acute cellular rejection (ACR). We present a rat osteomyocutaneous flap model using titrated tacrolimus administration that mimics the graft rejection patterns in our clinical hand transplant program. Comparison of outcomes in these models support a role for ischemia reperfusion injury (IRI) and microvascular changes in CR of skin and large-vessel vasculopathy. The potential of the surgical models for investigating mechanisms of rejection and vasculopathy in VCA and treatment interventions is presented. MATERIALS AND METHODS: Four rodent groups were evaluated: syngeneic controls (Group 1), allogeneic transient immunosuppression (Group 2), allogeneic suboptimal immunosuppression (Group 3), and allogeneic standard immunosuppression (Group 4). Animals were monitored for ACR, vasculopathy, and CR of the skin. RESULTS: Transient immunosuppression resulted in severe ACR within 2 wk of tacrolimus discontinuation. Standard immunosuppression resulted in minimal rejection but subclinical microvascular changes, including capillary thrombosis and luminal narrowing in arterioles in the donor skin. Further reduction in tacrolimus dose led to femoral vasculopathy and CR of the skin. Surprisingly, femoral vasculopathy was also observed in the syngeneic control group. CONCLUSIONS: Titration of tacrolimus in the allogeneic VCA model resulted in presentations of rejection and vasculopathy similar to those in patients and suggests vasculopathy starts at the microvascular level. This adjustable experimental model will allow the study of variables and interventions, such as external trauma or complement blockade, that may initiate or mitigate vasculopathy and CR in VCA.

International consensus recommendations on face transplantation: A 2-step Delphi study.

Face transplantation is a viable reconstructive approach for severe craniofacial defects. Despite the evolution witnessed in the field, ethical aspects, clinical and psychosocial implications, public perception, and economic sustainability remain the subject of debate and unanswered questions. Furthermore, poor data reporting and sharing, the absence of standardized metrics for outcome evaluation, and the lack of consensus definitions of success and failure have hampered the development of a "transplantation culture" on a global scale. We completed a 2-round online modified Delphi process with 35 international face transplant stakeholders, including surgeons, clinicians, psychologists, psychiatrists, ethicists, policymakers, and researchers, with a representation of 10 of the 19 face transplant teams that had already performed the procedure and 73% of face transplants. Themes addressed included patient assessment and selection, indications, social support networks, clinical framework, surgical considerations, data on patient progress and outcomes, definitions of success and failure, public image and perception, and financial sustainability. The presented recommendations are the product of a shared commitment of face transplant teams to foster the development of face transplantation and are aimed at providing a gold standard of practice and policy.

Novel cell-based strategies for immunomodulation in vascularized composite allotransplantation.

PURPOSE OF REVIEW: Vascularized composite allotransplantation (VCA) has become a clinical reality in the past two decades. However, its routine clinical applications are limited by the risk of acute rejection, and the side effects of the lifelong immunosuppression. Therefore, there is a need for new protocols to induce tolerance and extend VCA survival. Cell- based therapies have emerged as an attractive strategy for tolerance induction in VCA. This manuscript reviews the current strategies and applications of cell-based therapies for tolerance induction in VCA. RECENT FINDINGS: Cellular therapies, including the application of bone marrow cells (BMC), mesenchymal stem cells (MSC), adipose stem cells, regulatory T cells (Treg) cells, dendritic cells and donor recipient chimeric cells (DRCC) show promising potential as a strategy to induce tolerance in VCA. Ongoing basic science research aims to provide insights into the mechanisms of action, homing, functional specialization and standardization of these cellular therapies. Additionally, translational preclinical and clinical studies are underway, showing encouraging outcomes. SUMMARY: Cellular therapies hold great potential and are supported by preclinical studies and clinical trials demonstrating safety and efficacy. However, further research is needed to develop novel cell-based immunosuppressive protocol for VCA.

Defining chronic rejection in vascularized composite allografts - do we have reliable surrogates to look for?

PURPOSE OF REVIEW: Chronic rejection (CR) is a major threat in the field of vascularized composite tissue allografts (VCAs) as it causes graft dysfunction and usually graft loss. Unfortunately, knowledge of CR in VCA is incomplete because of the limited number of VCA recipients, the heterogeneous nature of VCAs and the short follow-up. RECENT FINDINGS: The diagnosis of CR in VCA has relied on clinical and pathological findings. Clinical changes include graft fibrosis, dyschromia and ischemic/necrotic ulcerations. Pathological changes primarily affect allograft vessels and manifest with graft vasculopathy (i.e. myo-intimal proliferation and luminal narrowing of allograft vessels, leading to graft ischemia). Attempts are made to diagnose CR with non- or minimally-invasive techniques, such as imaging studies (ultrasound biomicroscopy, functional magnetic resonance imaging) and serum biomarkers. These techniques provide interesting results and further insight into the mechanisms of CR in VCA. SUMMARY: The diagnosis of CR in VCA still relies mainly on clinicopathological graft alterations; unfortunately, these become overt rather late during the rejection process, when reversal of CR is problematic. More recent, minimally- or non-invasive techniques have provided encouraging results, but their usefulness in the diagnosis of CR requires further studies. These data highlight the paramount importance of CR prevention.

Methods of ex vivo analysis of tissue status in vascularized composite allografts.

Vascularized composite allotransplantation can improve quality of life and restore functionality. However, the complex tissue composition of vascularized composite allografts (VCAs) presents unique clinical challenges that increase the likelihood of transplant rejection. Under prolonged static cold storage, highly damage-susceptible tissues such as muscle and nerve undergo irreversible degradation that may render allografts non-functional. Skin-containing VCA elicits an immunogenic response that increases the risk of recipient allograft rejection. The development of quantitative metrics to evaluate VCAs prior to and following transplantation are key to mitigating allograft rejection. Correspondingly, a broad range of bioanalytical methods have emerged to assess the progression of VCA rejection and characterize transplantation outcomes. To consolidate the current range of relevant technologies and expand on potential for development, methods to evaluate ex vivo VCA status are herein reviewed and comparatively assessed. The use of implantable physiological status monitoring biochips, non-invasive bioimpedance monitoring to assess edema, and deep learning algorithms to fuse disparate inputs to stratify VCAs are identified.

What is needed to ensure long-term sustainability for the field of vascularized composite allotransplantation?

The field of vascularized composite allotransplantation (VCA) has demonstrated remarkable advances since its inception with some excellent long-term results in a variety of graft types. However, unlike solid organ transplantation, it has yet to become mainstream. We therefore discuss strategies on ensuring long-term sustainability by addressing continued clinical developments of VCA to improve the risk-to-benefit balance, importance of public support, improved policy and financial support, and need for a bridge to the future of transplant surgery. There has been headway on all fronts and collaboration among the VCA centers for centralization of data and incorporation of patient voices will be essential for continued progress.

Minimally and Non-invasive Approaches to Rejection Identification in Vascularized Composite Allotransplantation.

OBJECTIVE: Rejection is common and pernicious following Vascularized Composite Allotransplantation (VCA). Current monitoring and diagnostic modalities include the clinical exam which is subjective and biopsy with dermatohistopathologic Banff grading, which is subjective and invasive. We reviewed literature exploring non- and minimally invasive modalities for diagnosing and monitoring rejection (NIMMs) in VCA. METHODS: PubMed, Cochrane, and Embase databases were queried, 3125 unique articles were reviewed, yielding 26 included studies exploring 17 distinct NIMMs. Broadly, NIMMs involved Imaging, Liquid Biomarkers, Epidermal Sampling, Clinical Grading Scales, and Introduction of Additional Donor Tissue. RESULTS: Serum biomarkers including MMP3 and donor-derived microparticles rose with rejection onset. Epidermal sampling non-invasively enabled measurement of cytokine & gene expression profiles implicated in rejection. Both hold promise for monitoring. Clinical grading scales were useful diagnostically as was reflection confocal microscopy. Introducing additional donor tissue showed promise for preemptively identifying rejection but requires additional allograft tissue burden for the recipient. CONCLUSION: NIMMs have the potential to dramatically improve monitoring and diagnosis in VCA. Many modalities show promise however, additional research is needed and a multimodal algorithmic approach should be explored.

A Single-Center Retrospective Evaluation of Decision-Making and Factors Motivating Hand Transplant Candidates.

INTRODUCTION: Advancements in vascularized composite allotransplantation have made hand transplants possible for persons living with upper limb loss. Hand transplantation is not a life-saving procedure, but rather a quality-of-life enhancing procedure; hence the risk of morbidity and mortality must be weighed against improvements in function and appearance. This study explored the decision-making process of patients evaluated for hand transplantation. METHODS/APPROACH: A qualitative case series study using retrospective chart data of evaluations was conducted between January 1, 2011 and February 28, 2020. Notes were extracted and read by three reviewers. Each case was summarized noting similarities and differences. FINDINGS: Nine patients underwent evaluation. Eight were no longer under evaluation and did not receive transplant; one was still undergoing evaluation. Patient motivations for evaluation were dissatisfaction with prostheses or self-image, chronic pain, performing activities of daily living, occupation, burden placed on caregivers, and concerns about overuse of non-affected limbs. Patients chose not to pursue transplantation due to rehabilitation time, immunosuppression, alternative treatments, and social and financial challenges. The clinical team discontinued evaluations due to unmet evaluation requirements, medical contraindications, or treatment alternatives. Different modes of shared decision-making were present depending on the party most heavily featured in the charts as driving decisions. DISCUSSION: This was an examination of shared decision-making with hand transplant candidates who did not proceed to transplant. Reasons for choosing alternative strategies for management were multifactorial. Lessons learned regarding patient motivations and shared decision-making can inform future interventions to better support patients.

Vascularized Composite Allotransplantation of the Hand: A Systematic Review of Eligibility Criteria.

BACKGROUND: Hand transplantation (HT) has emerged as an intervention of last resort for those who endured amputation or irreparable loss of upper extremity function. However, because of the considerable effort required for allograft management and the risks of lifelong immunosuppression, patient eligibility is critical to treatment success. Thus, the objective of this article is to investigate the reported eligibility criteria of HT centers globally. METHODS: A systematic review of the HT literature was conducted according to Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines, using PubMed, Cochrane, Ovid/Medline, and Scopus. Program Web sites and clinicaltrials.gov entries were included where available. RESULTS: A total of 354 articles were reviewed, 101 of which met inclusion criteria. Furthermore, 10 patient-facing Web sites and 11 clinical trials were included. The most reported criteria related to the capacity to manage the allograft posttransplantation, including access to follow-up, insurance coverage, psychological stability, and history of medical compliance. Other factors related to the impact of immunosuppression, such as active pregnancy and patient immune status, were less emphasized. CONCLUSIONS: Because of the novelty of the field, eligibility criteria continue to evolve. While there is consensus on certain eligibility factors, other criteria diverge between programs, and very few factors were considered absolute contraindications. As the popularity of the field continues to grow, we encourage the development of consensus evidence-based eligibility criteria.

[Research progress of allogeneic abdominal wall transplantation].

Objective: To summarize the research progress of surgical technique and immunosuppressive regimen of abdominal wall vascularized composite allograft transplantation in animals and clinical practice. Methods: The literature on abdominal wall transplantation at home and abroad in recent years was extensively reviewed and analyzed. Results: This review includes animal and clinical studies. In animal studies, partial or total full-thickness abdominal wall transplantation models have been successfully established by researchers. Also, the use of thoracolumbar nerves has been described as an important method for functional reconstruction and prevention of long-term muscle atrophy in allogeneic abdominal wall transplantation. In clinical studies, researchers have utilized four revascularization techniques to perform abdominal wall transplantation, which has a high survival rate and a low incidence of complications. Conclusion: Abdominal wall allotransplantation is a critical reconstructive option for the difficulty closure of complex abdominal wall defects. Realizing the recanalization of the nerve in transplanted abdominal wall to the recipient is very important for the functional recovery of the allograft. The developments of similar research are beneficial for the progress of abdominal wall allotransplantation.

Novel Strategies in Transplantation: Genetic Engineering and Vascularized Composite Allotransplantation.

INTRODUCTION: Despite the clinical success in vascularized composite allotransplantation (VCA), systemic immunosuppression remains necessary to prevent allograft rejection. Even with potent immunosuppressive regimens (tacrolimus, mycophenolate mofetil, and steroids), most patients experience several rejection episodes, often within the same year. The risk of systemic side effects must constantly be weighed against the risk of under-immunosuppression and, thus, acute and chronic rejection. In this context, genomic editing has emerged as a potential tool to minimize the need for toxic immunosuppressive regimens and has gained attention in the fields of solid organ transplantation and xenotransplantation. This strategy may also be relevant for the future of VCA. METHODS: We discuss the topic of genetic engineering and review recent developments in this field that justify investigating tools such as clustered regularly interspaced short palindromic repeats/Cas9 in the context of VCA. RESULTS: We propose specific strategies for VCA based on the most recent gene expression data. This includes the well-known strategy of tolerance induction. Specifically, targeting the interaction between antigen-presenting cells and recipient-derived T cells by CD40 knockout may be effective. The novelty for VCA is a discovery that donor-derived T lymphocytes may play a special role in allograft rejection of facial transplants. We suggest targeting these cells prior to transplantation (e.g., by ex vivo perfusion of the transplant) by knocking out genes necessary for the long-term persistence of donor-derived immune cells in the allograft. CONCLUSION: Despite the demonstrated feasibility of VCA in recent years, continued improvements to immunomodulatory strategies using tools like clustered regularly interspaced short palindromic repeats/Cas9 could lead to the development of approaches that mitigate the limitations associated with rejection of this life-giving procedure.