Cesar Vera-Donoso

Therapeutic Potential of Human Adipose-Derived Stem Cells (ADSCs) from Cancer Patients: A Pilot Study

Mesenchymal stem cells from adipose tissue (ADSCs) are an important source of cells for regenerative medicine. The therapeutic effect of culture-expanded adipose derived stem cells has been shown; however, optimal xeno-free culture conditions remain to be determined. Cancer patients, specifically those undergoing invasive surgery, constitute a subgroup of patients who could benefit from autologous stem cell transplantation. Although regenerative potential of their ADSCs could be affected by the disease and/or treatment, we are not aware of any study that has evaluated the therapeutic potential of ADSCs isolated from cancer patients in reference to that of ADSCs derived from healthy subjects. Here we report that ADSCs isolated from subabdominal adipose tissue of patients with urological neoplasms yielded similar growth kinetics, presented equivalent mesenchymal surface markers and showed similar differentiation potential into distinct mesodermal cell lineages: adipocytes, chondroblasts and osteoblasts than ADSCs isolated from adipose tissue of age-matched non-oncogenic participants, all under xeno-free growth culture conditions. Molecular karyotyping of patient expanded ADSCs genomes showed no disease-related alterations indicating their safety. In addition, vesicles <100 nm identified as exosomes (EXOs) which may be at least partly responsible for the attributed therapeutic paracrine effects of the ADSCs were effectively isolated from ADSCs and showed equivalent miRNA content regardless they were derived from cancer patients or non-oncogenic participants indicating that the repair capabilities of xeno-free expanded ADSCs are not compromised by patient condition and therefore their xeno-free culture expanded ADSCs should be suitable for autologous stem cell transplantation in a clinical setting.

Vitrification of kidney precursors as a new source for organ transplantation.

Kidney transplantation from deceased or living human donors has been limited by donor availability as opposed to the increasing demand, and by the risk of allograft loss rejection and immunosuppressive therapy toxicity. In recent years, xenotransplantation of developed kidney precursor cells has offered a novel solution for the unlimited supply of human donor organs. Specifically, transplantation of kidney precursors in adult hosts showed that intact embryonic kidneys underwent maturation, exhibiting functional properties, and averted humoural rejection post-transplantation from non-immunosuppressed hosts. Even if supply and demand could be balanced using xenotransplants or lab-grown organs from regenerative medicine, the future of these treatments would still be compromised by the ability to physically distribute the organs to patients in need and to produce these products in a way that allows adequate inventory control and quality assurance. Kidney precursors originating from fifteen-day old rabbit embryos were vitrified using Cryotop® as a device and VM3 as vitrification solution. After 3 months of storage in liquid nitrogen, 18 kidney precursors were transplanted into non-immunosuppressed adult hosts by laparoscopy surgery. Twenty-one days after allotransplantation, 9 new kidneys were recovered. All the new kidneys recovered exhibited significant growth and mature glomeruli. Having achieved these encouraging results, we report, for the first time, that it is possible to create a long-term biobank of kidney precursors as an unlimited source of organs for transplantation, facilitating the inventory control and distribution of organs.

Trasplante laparoscópico de metanefros: un paso inicial al xenotrasplante renal

BACKGROUND Embryonic kidney xenotransplantation could represent a new solution to the scarcity of kidneys for transplantation. OBJECTIVE To determine the feasibility of allogeneic laparoscopic transplantation of metanephroi (M) in rabbits. MATERIAL AND METHOD Microscopic dissection was conducted to obtain metanephroi from 14-day-old (24M), 15-day-old (20M) and 16-day-old (26M) embryos. Using single-port abdominal laparoscopy, a spinal needle was inserted percutaneously, through which the metanephroi were deposited (using an epidural catheter) close to a patent blood vessel in the retroperitoneal fat. Seventy metanephroi were transplanted to 18 rabbits. Three weeks later, the animals were examined through open surgery. We compared the embryonic maturity, the morphometric variables of the metanephroi and the development rate of the transplanted metanephroi. RESULTS The lower time limit for the extraction of metanephroi from the rabbits was day 14. Three weeks after transplantation, only 3/24 14-day-old metanephroi grew at minimal expression (12.5%). In contrast, 10/20 (50%) 15-day-old and 12/26 (46.1%) 16-day-old metanephroi grew. These metanephroi had differentiated sufficiently for the glomeruli, proximal and distal tubules and collecting ducts to develop normally. We detected no relevant immunological changes in the peripheral blood. CONCLUSIONS We have described for the first time in the literature the allogeneic laparoscopic transplantation of metanephroi from embryos as a feasible and noninvasive technique. The recipients did not require immunosuppression.

Embryonic Organ Transplantation: The New Era of Xenotransplantation

Here, we review the recent advances towards the use of organs from embryonic donors, antecedent investigations, and the latest work from our own laboratory exploring the utility for transplantation of embryonic kidney as an organ replacement therapy. In addition, we have recently reported, for the first time, that it is possible to create a longterm biobank of kidney precursors as an unlimited source of organs for xenotransplan‐ tation, facilitating inventory control and the distribution of organs. Kidney transplantation from deceased or living human donors has been limited by donor availability as opposed to the increasing demand. Simultaneously, the risk of loss of graft by rejection or toxicity of immunosuppressive therapy exacerbates this organ shortage. In recent years, xenotransplantation of developing pancreas and kidney precursor cells has offered a novel solution for the unlimited supply of human donor organs. Specifical‐ ly, transplantation of kidney precursors in adult hosts showed that intact embryonic kidneys underwent maturation, exhibiting functional properties, and averted humoral rejection post-transplantation from non-immunosuppressed hosts. Organ primordia engraft, attract a host vasculature, and differentiate following transplantation to ectopic sites. Attempts have been made to exploit these characteristics to achieve clinically relevant endpoints for end-stage renal disease using animal models. We focused on two main points: (a) performing transplantation by a minimally invasive laparoscopic procedure and (b) creating a long-term biobank of kidney precursors, as an unlimited source of organs for transplantation, facilitating the inventory control and the distribution of organs. Because even if supply and demand could be balanced using xenotransplants or laboratory-grown organs from regenerative medicine, the future of these treatments would still be compromised by the ability to physically distribute the organs to patients in need and to produce these products in a way that allows adequate inventory control and quality assurance.

MP81-09 RECELLULARIZED ACELLULAR MATRIX VS NON-RECELULLARIZED ACELLULAR MATRIX IN A RAT MODEL FOR IN VIVO NEO-BLADDER REGENERATION

INTRODUCTION AND OBJECTIVES: The use of decellularized natural matrices to recover a functional bladder has been shown to be the most appropriate source to reconstruct this organ. Our goal was to compare evolution of recellularized acellular matrix vs non-recelullarized matrix implanted in a rat model. METHODS: All experimental procedures were performed into consideration the ethical processes described by the animal committee of the center. Sprague-Dawley rats were used for the experiment. Here we compared the efficient and functional integration of a recellularized acellular matrix, previously seeded in vitro with human adipose tissue derived mesenchymal stem cells with a non-recelullarized matrix in a rat model of partial cystectomy and bladder substitution. RESULTS: Relevant and significant anatomical differences were found few days after neo bladders implantation. The human ADSC containing matrix showed a significant recovery of mature urothelium measured as the positive reactivity to p63 at the transitional epithelium layers. The expression of the mesoderm marker, cytokeratin 7 was also significantly induced within the regenerated connective tissue. Interestingly a higher expression of smooth muscle actin was found at both, the internal and peripheral transversal and longitudinal smooth muscle layers in the hADSC-recellularized matrix. However, no significant induction in any of the transplanted groups, in comparison with native bladder, was found for CD31 expression, endothelium marker. The presence of the hADSC, by most probably the paracrine release of proregenerative factors, favors a faster and more efficient endogenous regeneration of the ectopic bladder allowing a faster voluntary urodynamic recovery within a more specialized an mature tissue. CONCLUSIONS: Although bladder recellularization and urodynamic functional recovery have been successfully tested in small animals, and here we show the relevant benefits of the use of human ADSC on this regeneration process, more efficient procedures ex-vivo with a better understanding of the mechanisms involved of the ectopic transplanted cells is required in order to improve the tissue maturation in terms of peripheral nerve innervation and vascularization for a proper clinical translation

Bioengineering Approaches for Bladder Regeneration

Current clinical strategies for bladder reconstruction or substitution are associated to serious problems. Therefore, new alternative approaches are becoming more and more necessary. The purpose of this work is to review the state of the art of the current bioengineering advances and obstacles reported in bladder regeneration. Tissue bladder engineering requires an ideal engineered bladder scaffold composed of a biocompatible material suitable to sustain the mechanical forces necessary for bladder filling and emptying. In addition, an engineered bladder needs to reconstruct a compliant muscular wall and a highly specialized urothelium, well-orchestrated under control of autonomic and sensory innervations. Bioreactors play a very important role allowing cell growth and specialization into a tissue-engineered vascular construct within a physiological environment. Bioprinting technology is rapidly progressing, achieving the generation of custom-made structural supports using an increasing number of different polymers as ink with a high capacity of reproducibility. Although many promising results have been achieved, few of them have been tested with clinical success. This lack of satisfactory applications is a good reason to discourage researchers in this field and explains, somehow, the limited high-impact scientific production in this area during the last decade, emphasizing that still much more progress is required before bioengineered bladders become a commonplace in the clinical setting.

Impact of lymph node dissection at the time of radical nephrectomy with tumor thrombectomy on oncological outcomes: Results from the International Renal Cell Carcinoma-Venous Thrombus Consortium (IRCC-VTC)

OBJECTIVES To study the effect of lymph node dissection (LND) at the time of nephrectomy and tumor thrombectomy on oncological outcomes in patients with renal cell carcinoma (RCC) and tumor thrombus. PATIENTS AND METHODS The records of 1,978 patients with RCC and tumor thrombus who underwent radical nephrectomy and tumor thrombectomy from 1985 to 2014 at 24 centers were analyzed. None of the patients had distant metastases. Extent and pathologic results of LND were compared with respect to cancer-specific survival (CSS). Multivariable Cox regression models were used to quantify the effect of multiple covariates. RESULTS LND was performed in 1,026 patients. In multivariable analysis, the presence of LN metastasis, the number of positive LNs, and LN density were independently associated with cancer-specific mortality (CSM). Clinical node-negative (cN-) disease was documented in 573 patients, 447 of them underwent LND with 43 cN- patients (9.6%) revealing positive LNs at pathology. LN positive cN- patients showed significantly better CSS when compared to LN positive cN+ patients. In multivariable analysis, positive cN status in LN positive patients was a significant predictor of CSM (HR, 2.923; P = 0.015). CONCLUSIONS The number of positive nodes harvested during LND and LN density was strong prognostic indicators of CSS, while number of removed LNs did not have a significant effect on CSS. The rate of pN1 patients among clinically node-negative patients was relatively high, and LND in these patients suggested a survival benefit. However, only a randomized trial can determine the absolute benefit of LND in this setting.