Adrenocortical cell transplantation in scid mice: the role of the host animals' adrenal glands

Adrenocortical cell transplantation is a powerful technique for the investigation of the regulation of adrenocortical structure and function. Some classical organ and tissue transplantation experiments suggest that the success of transplantation depends on the activity of the pituitary gland and other endocrine systems, and is therefore influenced by the host animals’ own adrenal glands. For this reason, our experiments have usually been performed on adrenalectomized animals. However, we show here that cell transplantation experiments, involving the introduction of bovine adrenocortical cells into scid mice, do produce transplant tissues in the presence of the host animals’ adrenal glands. However, the tissue that forms is small and its cells also smaller than usual. When the adrenals of such animals are removed in a second surgical procedure, the transplants show a rapid increase in steroidogenic function and a slower increase in size, over several weeks. We conclude that the initial process by which transplanted adrenocortical cells organize into a tissue structure is not affected by the presence of the host animals’ adrenal glands, but the growth of the transplants is limited until the adrenal glands are removed.     

Adrenocortical cell transplantation in scid mice: the role of the host animals’ adrenal glands

Adrenocortical cell transplantation is a powerful technique for the investigation of the regulation of adrenocortical structure and function. Some classical organ and tissue transplantation experiments suggest that the success of transplantation depends on the activity of the pituitary gland and other endocrine systems, and is therefore influenced by the host animals’ own adrenal glands. For this reason, our experiments have usually been performed on adrenalectomized animals. However, we show here that cell transplantation experiments, involving the introduction of bovine adrenocortical cells into scid mice, do produce transplant tissues in the presence of the host animals’ adrenal glands. However, the tissue that forms is small and its cells also smaller than usual. When the adrenals of such animals are removed in a second surgical procedure, the transplants show a rapid increase in steroidogenic function and a slower increase in size, over several weeks. We conclude that the initial process by which transplanted adrenocortical cells organize into a tissue structure is not affected by the presence of the host animals’ adrenal glands, but the growth of the transplants is limited until the adrenal glands are removed.     

Macrophage-neoplastic cell interactions: implications for neoplastic cell growth

Abstract Subcutaneous transplantation of EL4 lymphoma cells within C57BL10 mice evoked an oedematous inflammatory responses involving increased leukopoiesis within the bone marrow, a blood leukocytosis, an influx of leukocytes into the transplant and surrounding host connective tissues, and extensive remodelling of sorrounding host connective tissues invloving fibroplasia and angiogenesis. Dexamethasone not only significantly reduced the numbers of circulating blood leukocytes within C57BL10 mice bearing the subcutaneous EL4 lymphoma transplants, but also reduced the oedematous inflammatory response to the transplants. The decreased influx of inflammatory leukocytes into a site of EL4 lymphoma cell transplantation within the dexamethasone-treated mice, was accompanied by reduced growth of the transplants. Although the EL4 lymphoma cells produce factors with Colony Stimulating Factor activity and with chemotactic activity for cells of the monocyte-macrophage lineage, they do not appear to produce fibroblast growth factors directly but can induce (or stimulate) macrophages to generate fibroblast growth factors in vitro. While not directly inhibiting the growth of subcutaneous fibroblast in vitro, dexamethasone does suppress the production and/or activity of fibroblast factors generated through macrophage-EL4 cell interactions in vitro. The inhibitory effects of dexamethasone on macrophage influx, fibroplasia and angiogenesis within the connective tissue sorrounding the EL4 lymphoma transplants appear to be casually related events and would account for the inhibitory effect of dexamethasone on the growth of the lymphoma transplants.     

Conjunctival reconstruction with progenitor cell-derived autologous epidermal sheets in rhesus monkey

Severe ocular surface diseases are some of the most challenging problems that the clinician faces today. Conventional management is generally unsatisfactory, and the long-term ocular consequences of these conditions are devastating. It is significantly important to find a substitute for conjunctival epithelial cells. This study was to explore the possibility of progenitor cell-derived epidermal sheets on denuded amniotic membrane to reconstruct ocular surface of conjunctiva damaged monkeys. We isolated epidermal progenitor cells of rhesus monkeys by type IV collagen adhesion, and then expanded progenitor cell-derived epidermal sheets on denuded amniotic membrane ex vivo. At 3 weeks after the conjunctiva injury, the damaged ocular surface of four monkeys was surgically reconstructed by transplanting the autologous cultivated epidermal progenitor cells. At 2 weeks after surgery, transplants were removed and examined with Hematoxylin-eosin staining, Periodic acid Schiff staining, immunofluorescent staining, scanning and transmission electron microscopy. Histological examination of transplanted sheets revealed that the cell sheets were healthy alive, adhered well to the denuded amniotic membrane, and had several layers of epithelial cells. Electron microscopy showed that the epithelial cells were very similar in appearance to those of normal conjunctival epithelium, even without goblet cell detected. Epithelial cells of transplants had numerous desmosomal junctions and were attached to the amniotic membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the conjunctival specific markers, mucin 4 and keratin 4, in the transplanted epidermal progenitor cells. In conclusion, our present study successfully reconstructed conjunctiva with autologous transplantation of progenitor cell-derived epidermal sheets on denuded AM in conjunctival damaged monkeys, which is the first step toward assessing the use of autologous transplantation of progenitor cells of nonocular surface origin. Epidermal progenitor cells could be provided as a new substitute for conjunctival epithelial cells to overcome the problems of autologous conjunctiva shortage.     

Bone Allografts: Past, Present and Future

Bone allograft transplantation has been performed in humans for more than one hundred and twenty years. During the first one hundred years (1880–1980), the major problem in bone allograft transplantation was availability. Most of the bone grafts used during this time were autografts. Allografts were not available due to a lack of legislation protecting procurers and processers. In addition, surgical procedures requiring allografts were not being performed. During the next twenty years (1980–2000), as allografis began to be used, the major issue was safety. Diseases transmitted during this period included AIDS and hepatitis. Avoidance of disease transmission became paramount. Sensitive blood tests and extensive efforts by bone banks to develop ways to clean. bone and clear it of infectious agents helped provide safe transplants. With concerns of availability and safety receding, the major issue in the future (2000–? ) will be the efficacy of the transplant. How allograft bone remodels in the host, how it incorporates and heals to host bone and how it integrates with the host skeleton will be the most important concerns of bone bankers and tissue transplant surgeons. Future research efforts will be applied to bone allograft transplantation to ensure that bone transplants heal quickly and sufficiently to be able to function as part of the weight-bearing skeletal system.     

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants.

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.     

Application of a cell sheet–polymer film complex with temperature sensitivity for increased mechanical strength and cell alignment capability

We have succeeded in fabricating a cell sheet–polymer film complex involving a temperature‐sensitive polymer that has enough mechanical strength that can be manipulated even by forceps. The polymer film can be removed by lowering the temperature after transplantation, demonstrating its potential use in regenerative medicine. Recently, tissue engineering involving cell sheets was developed, tissues being fabricated by layering of these cell sheets. This technique promises high density cell packing, which is important for native cell functions, and successful heart therapy using cardiac cell sheets has been reported. On the other hand, the fabrication of a large tissue using cell sheets is difficult because of fragility of the cell sheets. Here, we have developed a novel method in which cells are attached to a temperature‐sensitive poly‐N‐isopropylacrylamide film mixed with laminin and collagen IV, and report that the cell sheet–polymer film complex can be manipulated with forceps. A cell sheet can be removed from the polymer film by lowering the temperature after the manipulation. We have utilized this technique for the primary myocardium and fabricated a physiologically active multi‐layered cardiac cell sheet. By applying a micropattern to this polymer film, we have succeeded in making a skeletal muscle cell sheet in which myotubes are oriented in the desired direction. Overall, we showed that this method is useful for cell sheet manipulation, morphogenesis, and transplantation. Biotechnol. Bioeng. 2009;103: 370–377. © 2009 Wiley Periodicals, Inc.     

The Fate of Myoblasts Following Transplantation into Mature Muscle

Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme β-galactosidase (β-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (β-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and/β-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest β-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell trans-plantation and for the application of such studies to cell therapy and cell-mediated gene therapy.     

Donation transplants and tissue banking in Mexico

Knowledge about transplants in Mexico goes back to the Aztec period.Today the need for organ and tissue transplants in Mexico is high; theestimatednumber is 100,000 patients, but there are only 2 donors per million population,for corneas. The organ, tissue and cell transplantation law which was modifiedin 2,000, establishes that when a person dies, he will be a potential donor oforgans and tissues. This new law will give hope to many patients, since it isexpected to increase significantly the amount of organs and tissues fortransplants. At present Mexico has 178 hospitals that are authorized to carryout organ and tissue transplants, and 53 Tissue Banks.     

Myocardial tissue engineering: toward a bioartificial pump

Regenerative therapies, including cell injection and bioengineered tissue transplantation, have the potential to treat severe heart failure. Direct implantation of isolated skeletal myoblasts and bone-marrow-derived cells has already been clinically performed and research on fabricating three-dimensional (3-D) cardiac grafts using tissue engineering technologies has also now been initiated. In contrast to conventional scaffold-based methods, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cell sheets to construct 3-D cell-dense tissues. Upon layering, individual cardiac cell sheets integrate to form a single, continuous, cell-dense tissue that resembles native cardiac tissue. The transplantation of layered cardiac cell sheets is able to repair damaged hearts. As the next step, we have attempted to promote neovascularization within bioengineered myocardial tissues to overcome the longstanding limitations of engineered tissue thickness. Finally, as a possible advanced therapy, we are now trying to fabricate functional myocardial tubes that may have a potential for circulatory support. Cell sheet-based tissue engineering technologies therefore show an enormous promise as a novel approach in the field of myocardial tissue engineering.     

Cryopreservation of embryonic cerebral tissue of rat.

Embryonic cerebral tissues (ECT) either fresh or frozen-stored, were cultured and transplanted into the cerebella of neonatal host rats. Many variables including composition of the freezing medium, freezing and thawing rates, and storage time in liquid nitrogen were studied systematically. The results indicated that the following conditions yielded good results for tissue culture: using 1 M Me2SO as the cryoprotectant, freezing the brain tissues at a rate of 1 degrees C/min until it reached -70 degrees C, storing the frozen samples in liquid nitrogen and thawing them fast in a 37 degrees C water bath. The viability of the frozen-thawed tissues was assessed by their abilities to grow and differentiate in vitro and in vivo after intracerebral grafting. In tissue culture, growth and differentiation were similar to those of the fresh ECT. Cell morphology and staining reactions were normal in supravital methylene blue staining, cresyl violet staining, and acetylcholinesterase staining. Neurons had well-developed Nissl bodies, and cholinergic neurons also differentiated. Autoradiographic studies showed that more than 50% of the neurons had the ability to uptake gamma-aminobutyric acid with high affinity. In brain tissue transplantation, 9 of 12 transplants survived subsequent grafting after cryopreservation. Moreover, the grafts of surviving cryopreserved tissue displayed cytological and cytoarchitectural characteristics identical to those of fresh grafts. All grafts were integrated with the surrounding host neural tissue. This suggested that there may be synaptic connections between the transplants and the host brain tissues. From this and similar studies on the subject by others wer conclude that cryopreservation is a feasible method for storage of embryonic brain tissue to be used later for intracerebral grafting.     

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.     

Pancreatic transplant surgery and stem cell therapy: Finding the balance between therapeutic advances and ethical principles

The latest achievements in the field of pancreas transplantation and stem cell therapy require an effort by the scientific community to clarify the ethical implications of pioneering treatments, often characterized by high complexity from a surgical point of view, due to transplantation of multiple organs at the same time or at different times, and from an immunological point of view for stem cell therapy. The fundamental value in the field of organ transplants is, of course, a solidarity principle, namely that of protecting the health and life of people for whom transplantation is a condition of functional recovery, or even of survival. The nature of this value is that of a concept to which the legal discipline of transplants entrusts its own ethical dignity and for which it has ensured a constitutional recognition in different systems. The general principle of respect for human life, both of the donor and of the recipient, evokes the need not to put oneself and one’s neighbor in dangerous conditions. The present ethical reflection aims to find a balance between the latest therapeutic advances and several concepts including the idea of the person, the respect due to the dead, the voluntary nature of the donation and the consent to the same, the gratuitousness of the donation, the scientific progress and the development of surgical techniques, and the policies of health promotion.     

Meiosis in autologous ectopic transplants of immature testicular tissue grafted to Callithrix jacchus

Grafting of immature testicular tissue provides a tool to examine testicular development and may offer a perspective for preservation of fertility in prepubertal patients. Successful xenografting in mice, resulting in mature spermatids, has been performed in several species but has failed with testicular tissues from the common marmoset, Callithrix jacchus. Previous data indicate that the hormonal milieu provided by the mouse host might cause this failure. We conducted autologous ectopic transplantation of testicular fragments under the back skin in newborn marmoset monkeys. Seventeen months after transplantation, we found viable transplants in 2 out of the 4 grafted animals. In the transplants, tubules developed up to a state intermediate between the pregraft situation and adult controls. Dividing spermatogonia and primary spermatocytes were present. Boule-like positivity and CDC25A negativity indicated that spermatogenesis was arrested at early meiosis. Immunohistochemistry revealed normal maturation of Sertoli cells, Leydig cells, and peritubular cells. Serum testosterone values were not restored to the normal range and bioactive chorionic gonadotropin levels increased to castrate levels. Meiotic arrest could have occurred in the grafts because of lack of sufficient testosterone or because of hyperthermia caused by the ectopic position of the grafts. We conclude that autologous transplants of immature testicular tissues in the marmoset can mature up to meiosis but that normal serum testosterone levels are not restored. Further studies have to be performed to overcome the meiotic arrest to explore the model further and to develop therapeutic options.     

Single cell transplantation reveals interspecific cell communication in Drosophila chimeras

Cell-cell communication is not only a common strategy for cell fate specification in vertebrates, but plays important roles in invertebrate development as well. We report here on experiments testing the compatibility of mechanisms specifying cell fate among six different Drosophila species. Following interspecific transplantation, the development of single ectodermal cells was traced in order to test their abilities to proliferate and differentiate in a heterologous environment. Despite considerable differences in cell size and length of cell cycle among some of the species, the transplants gave rise to fully differentiated clones that were integrated into the host tissue. Clones comprised cells of epidermal and/or neural histotypes, indicating that mechanisms mediating the epidermal/neural dichotomy in the ectoderm are conserved between the species. Cells of the neural lineages differentiated into neurones, glia, or both. Moreover, heterologous neurones sent out axons that followed major pathways along nerves and within the neuropile, demonstrating their ability to recognize positional cues in the heterologous CNS of the host.     

Host NKT cells can prevent graft-versus-host disease and permit graft antitumor activity after bone marrow transplantation

Allogeneic bone marrow transplantation is a curative treatment for leukemia and lymphoma, but graft-vs-host disease (GVHD) remains a major complication. Using a GVHD protective nonmyeloablative conditioning regimen of total lymphoid irradiation and antithymocyte serum (TLI/ATS) in mice that has been recently adapted to clinical studies, we show that regulatory host NKT cells prevent the expansion and tissue inflammation induced by donor T cells, but allow retention of the killing activity of donor T cells against the BCL1 B cell lymphoma. Whereas wild-type hosts given transplants from wild-type donors were protected against progressive tumor growth and lethal GVHD, NKT cell-deficient CD1d-/- and Jalpha-18-/- host mice given wild-type transplants cleared the tumor cells but died of GVHD. In contrast, wild-type hosts given transplants from CD8-/- or perforin-/- donors had progressive tumor growth without GVHD. Injection of host-type NKT cells into Jalpha-18-/- host mice conditioned with TLI/ATS markedly reduced the early expansion and colon injury induced by donor T cells. In conclusion, after TLI/ATS host conditioning and allogeneic bone marrow transplantation, host NKT cells can separate the proinflammatory and tumor cytolytic functions of donor T cells.     

Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.     

Autologous Transplantation of Oral Mucosal Epithelial Cell Sheets Cultured on an Amniotic Membrane Substrate for Intraoral Mucosal Defects

The human amniotic membrane (AM) is a thin intrauterine placental membrane that is highly biocompatible and possesses anti-inflammatory and anti-scarring properties. Using AM, we developed a novel method for cultivating oral mucosal epithelial cell sheets. We investigated the autologous transplantation of oral mucosal epithelial cells cultured on AM in patients undergoing oral surgeries. We obtained specimens of AM from women undergoing cesarean sections. This study included five patients without any history of a medical disorder who underwent autologous cultured oral epithelial transplantation following oral surgical procedures. Using oral mucosal biopsy specimens obtained from these patients, we cultured oral epithelial cells on an AM carrier. We transplanted the resultant cell sheets onto the oral mucosal defects. Patients were followed-up for at least 12 months after transplantation. After 2–3 weeks of being cultured on AM, epithelial cells were well differentiated and had stratified into five to seven layers. Immunohistochemistry revealed that the cultured cells expressed highly specific mucosal epithelial cell markers and basement membrane proteins. After the surgical procedures, no infection, bleeding, rejection, or sheet detachment occurred at the reconstructed sites, at which new oral mucous membranes were evident. No recurrence was observed in the long-term follow-up, and the postoperative course was excellent. Our results suggest that AM-cultured oral mucosal epithelial cell sheets represent a useful biomaterial and feasible method for oral mucosal reconstruction. However, our primary clinical study only evaluated their effects on a limited number of small oral mucosal defects.     

Adipose‐derived stem cell spheroids are superior to single‐cell suspensions to improve fat autograft long‐term survival

Autologous fat transplantation is a widely used procedure for surgical reconstruction of tissues. The resorption rate of this transplantation remains high and unpredictable, reinforcing the need of adjuvant treatments that increase the long‐term stability of grafts. Adipose‐derived stem cells (ASC) introduced as single cells in fat has been shown clinically to reduce the resorption of fat grafts. On the other hand, the formulation of ASC into cell spheroids results in the enhancement of their regenerative potential. In this study, we developed a novel method to produce highly homogeneous ASC spheroids and characterized their features and efficacy on fat transplantation. Spheroids conserved ASC markers and multipotency. A regenerative gene expression profile was maintained, and genes linked to autophagy were upregulated whereas proliferation was decreased. Their secreted proteome was enriched in comparison with single‐cell ASC suspension. Addition of spheroids to fat graft in an animal model of transplantation resulted in a better graft long‐term stability when compared to single ASC suspension. In conclusion, we provide a novel method to manufacture homogenous ASC spheroids. These ASC spheroids are superior to ASC in single‐cell suspension to improve the stability of fat transplants, reinforcing their potential in reconstructive surgery.     

Correction of the cornrow hair transplant and other common problems in surgical hair restoration

Hair on a man's head is an important emblem of health, youth, and vitality. As in all areas of cosmetic surgery, the refinements of surgical technique and instrumentation have improved the results of hair transplantation. The state of the art in hair grafting today produces a result that is undetectable as being a surgical hair transplant. Many earlier techniques of plug hair transplantation are not aesthetically acceptable by today's standards. This is especially true in the face of progressive hair loss, which can unmask previously camouflaged cornrow plugs. A technique to reduce the plugs and recycle the grafts into smaller grafts is described. The recycled hair grafts can be combined with scalp lifting, scalp reductions, and occipital harvesting of grafts to improve the results of cornrow appearing hair transplants and other problems of surgical hair restoration.