Pancreas and Kidney Transplantation Using Embryonic Donor Organs

One novel solution to the shortage of human organs available for transplantation envisions ‘growing’ new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. We and others have shown that renal anlagen (metanephroi) transplanted into animal hosts undergo differentiation and growth, become vascularized by blood vessels of host origin and exhibit excretory function. Metanephroi can be stored for up to 3 days in vitro prior to transplantation with no impairment in growth or function post-implantation. Metanephroi can be transplanted across both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. Similarly, pancreatic anlagen can be transplanted across concordant and highly disparate barriers, and undergo growth, differentiation and secrete insulin in a physiological manner following intra-peritoneal placement. Implantation of the embryonic pancreas, is followed by selective differentiation of islet components. Here we review studies exploring the potential therapeutic applicability for organogenesis of the kidney or endocrine pancreas.     

Organogenesis of Kidney and Endocrine Pancreas

Growing new organs in situ by implanting developing animal organ primordia (organogenesis) represents a novel solution to the problem of limited supply for human donor organs that offers advantages relative to transplanting embryonic stem (ES) cells or xenotransplantation of developed organs. Successful transplantation of organ primordia depends on obtaining them at defined windows during embryonic development within which the risk of teratogenicity is eliminated, growth potential is maximized, and immunogenicity is reduced. We and others have shown that renal primordia transplanted into the mesentery undergo differentiation and growth, become vascularized by blood vessels of host origin, exhibit excretory function and support life in otherwise anephric hosts. Renal primordia can be transplanted across isogeneic, allogeneic or xenogeneic barriers. Pancreatic primordia can be transplanted across the same barriers undergo growth, and differentiation of endocrine components only and secrete insulin in a physiological manner following mesenteric placement. Insulin-secreting cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin-diabetic (type 1) Lewis rats or ZDF diabetic (type 2) rats or STZ-diabetic rhesus macaques engraft without the need for host immune-suppression. Our findings in diabetic macaques represent the first steps in the opening of a window for a novel treatment of diabetes in humans.     

HL-A and Kidney Transplantation

AT present the role of HL-A antigens in the rejection of organ transplants is uncertain; we therefore wish to report the results of renal transplantations, under known HL-A compatibility conditions, performed in France since 1959. The 179 cases already published² have now increased to 416 cases³ including 84 sibling (SS), parent-to-child (PC) and 253 unrelated (UR) allografts. Only three cases were excluded from the analysis, which was similar to that previously used². We calculated the probability of incompatibility for the non-determined HL-A antigens, assuming the presence of four HL-A antigens of similar strength in each individual. Differences between antigens having serological similarities (cross-reactions) were considered in a first analysis as incompatibilities and in a second analysis as identities.     

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells ¹. The whole embryonic organ can be cultured at multiple stages of development ^2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.     

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels¹. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation². A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo^3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ^2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing β-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium^7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.     

Orthotopic Kidney Transplantation in Mice: Technique Using Cuff for Renal Vein Anastomosis

Mouse renal transplantation is a technically challenging procedure. Although the first kidney transplants in mice were performed over 34 years ago and refined some years later, the classical techniques of mouse renal transplantation required clamping both vena cava and aorta simultaneously and carry out suture anastomoses of the renal artery and vein in a heterotopic position. In our laboratory, we have successfully developed mouse orthotopic kidney transplantation for the first time, using a rapid “cuffed” renal vein technique for vessel anastomosis, wherein the donor’s renal vein was inserted through an intravenous catheter, folded back and tied. During grafting, the cuffed renal vein was directly inserted into the recipient’s renal vein without the need for the clamping vena cava and suturing of renal vein. This technique allowed for the exact transplantation of the kidney into the original position, compared to the classical technique, and has significantly shortened the clamping time due to a quicker and precise anastomosis of renal vein as described. This also allowed for a quicker recovery of the lower extremity activity, reduction in myoglobinuria with resultant kidney graft survival of 88.9%. Thus we believe that the cuffed renal vein technique simplifies microvascular anastomoses and affords important additional benefits.     

中华鳖胚胎肾脏的发育

采用H.E常规组织学方法对92例中华鳖Pelodiscus sinensis胚胎发育过程中肾脏的发育做了研究.结果 表明:5 d前仅见由前肾管和前肾导管构成的前肾, 5～12 d是中肾的发育期,由中肾管、中肾小管和肾小体构成,12 d前后中肾逐步退化,后肾原基形成,16 d后肾各部分开始分化.     

Donor Safety in Living Donor Liver Transplantation: A Single-Center Analysis of 300 Cases

Aim

To evaluate the safety to donors of living-donor liver transplantation.

Methods

This study included 300 consecutive living liver tissue donors who underwent operations at our center from July 2002 to December 2012. We evaluated the safety of donors with regard to three aspects complications were recorded prospectively and stratified by grade according to Clavien’s classification, and the data were compared in two stages (the first 5 years’ experience (pre-January 2008) and the latter 5 years’ experience (post-January 2008); laboratory tests such as liver function and blood biochemistry were performed; and the health-related quality of life was evaluated.

Results

There was no donor mortality at our center, and the overall morbidity rate was 25.3%. Most of the complications of living donors were either grade I or II. There were significantly fewer complications in the latter period of our study than in the initial period (19.9% vs 32.6%, P<0.001), and biliary complications were the most common complications, with an incidence of 9%. All of the liver dysfunction was temporary; however, the post-operative suppression of platelet count lasted for years. Although within the normal range, eight years after operation, 22 donors showed lower platelet levels (189×10⁹/L) compared with the pre-operative levels (267×10⁹/L) (P<0.05). A total of 98.4% of donors had returned to their previous levels of social activity and work, and 99.2% of donors would donate again if it was required and feasible. With the exception of two donors who experienced grade III complications (whose recipients died) and a few cases of abdominal discomfort, fatigue, chronic pain and scar itching, none of the living donors were affected by physical problems.

Conclusion

With careful donor selection and specialized patient care, low morbidity rates and satisfactory long-term recovery can be achieved after hepatectomy for living-donor liver transplantation.     

大鼠胰腺胚胎发育功能相关基因表达谱的分析

目的:研究大鼠胰腺胚胎发育不同阶段的基因表达谱,对比其功能相关基因随大鼠胰腺发育的变化.方法:采用显微分离及提取技术获得胚胎发育不同阶段胰腺组织并提取RNA,采用高密度寡核普酸芯片(Affemetrix芯片)对胚胎发育至第12.5天、15.5天、18.5天胚胎胰腺及成年胰腺进行基因转录水平分析,用生物信息学方法分析具体基因的表达情况.结果:胰腺的生物学功能尤其beta细胞功能相关基因insulin RNA,amylopsin RNA,GLUT-2 RNA等在胚胎15.5及18.5天显著高表达.结论:E15.5到E18.5直至出生是胰腺功能完善和成熟的阶段,这个时期以细胞功能成熟为主.     

胚胎干细胞移植治疗帕金森病

胚胎干细胞起源于植入前胚胎的内细胞团(inner cell mass,ICM),能够在体外进行增殖并能分化成三个胚层的所有细胞类型.如果能有效地将胚胎干细胞(embryonic stem cell,ES细胞)诱导分化为特定的细胞类型,ES细胞将成为细胞替代治疗中供体细胞的一个理想来源,从而可以应用于帕金森病(Parkinson's disease,PD)、糖尿病、心脓死等的退行性病变疾病的治疗.近年来,将ES细胞来源的多巴胺能神经元用于PD治疗的研究越来越广泛.现对这些研究中有关基因表达调控、实验研究进展、临床应用以及所遇到的问题作一综述.虽然这些研究还没能最终大面积应用于临床.但为PD的治疗提供了一个广阔的应用前景.     

Gut Microbiota and Tacrolimus Dosing in Kidney Transplantation

Tacrolimus dosing to establish therapeutic levels in recipients of organ transplants is a challenging task because of much interpatient and intrapatient variability in drug absorption, metabolism, and disposition. In view of the reported impact of gut microbial species on drug metabolism, we investigated the relationship between the gut microbiota and tacrolimus dosing requirements in this pilot study of adult kidney transplant recipients. Serial fecal specimens were collected during the first month of transplantation from 19 kidney transplant recipients who either required a 50% increase from initial tacrolimus dosing during the first month of transplantation (Dose Escalation Group, n=5) or did not require such an increase (Dose Stable Group, n=14). We characterized bacterial composition in the fecal specimens by deep sequencing of the PCR amplified 16S rRNA V4-V5 region and we investigated the hypothesis that gut microbial composition is associated with tacrolimus dosing requirements. Initial tacrolimus dosing was similar in the Dose Escalation Group and in the Stable Group (4.2±1.1 mg/day vs. 3.8±0.8 mg/day, respectively, P=0.61, two-way between-group ANOVA using contrasts) but became higher in the Dose Escalation Group than in the Dose Stable Group by the end of the first transplantation month (9.6±2.4 mg/day vs. 3.3±1.5 mg/day, respectively, P<0.001). Our systematic characterization of the gut microbial composition identified that fecal Faecalibacterium prausnitzii abundance in the first week of transplantation was 11.8% in the Dose Escalation Group and 0.8% in the Dose Stable Group (P=0.002, Wilcoxon Rank Sum test, P<0.05 after Benjamini-Hochberg correction for multiple hypotheses). Fecal Faecalibacterium prausnitzii abundance in the first week of transplantation was positively correlated with future tacrolimus dosing at 1 month (R=0.57, P=0.01) and had a coefficient±standard error of 1.0±0.6 (P=0.08) after multivariable linear regression. Our novel observations may help further explain inter-individual differences in tacrolimus dosing to achieve therapeutic levels.     

Fetal Tissue Banking for Transplantation: Characteristics of the Donor Population and Considerations for Donor and Tissue Screening

We initiated this study to evaluate the suitability for therapeutic use in transplantation of tissues obtained from human abortuses. We have developed protocols for the collection, handling and preservation of hepatic stem cells from electively aborted embryos and have developed methods for assessment of the cells so derived and processed. In this paper we present our findings regarding screening of potential donors, acquisition of fetal tissues, and assessment of the tissues for potentially infectious contaminants. We assess the suitability of the tissue donors according to current standards used for donors of commonly transplanted tissues (e.g., bone grafts, skin grafts and heart valves) and present data regarding the real availability of tissues from elective abortion procedures that would meet those standard tissue banking criteria.We specifically evaluated the donor's willingness to provide a blood sample for testing, conducted a detailed interview similar to those used for typical organ and tissue donors, and assessed the type and incidence of contamination in collected tissues. We find that although many women are willing to consent to use of the tissues for transplantation, attrition from the study for various reasons results in few fetal organs ultimately realistically available for transplantation. Typical reasons for attrition include: unwillingness to have a blood sample drawn or tested, positive serology results, social/medical high risk factors for acquisition of transmissible disease, no identifiable organs available, and unacceptable microbial contamination. Thus, although it might seem that due to the numbers of abortions performed annually, that there would be substantial numbers of suitable tissues available, only a small proportion are truly suitable for transplantation.