Generation of normal progeny by intracytoplasmic sperm injection following grafting of testicular tissue from cloned mice that died postnatally

Animal cloning by nuclear transfer has been successful in several species and was expected to become an alternative reproductive technique. Among the problems associated with this cloning technique, however, are its low success rate and high mortality of cloned animals even if they develop to term. Nuclear transfer has thus come to be considered too difficult to apply as a reproductive technique. The transplantation of male germ cells or pieces of testicular tissue has enabled the induction of spermatogenesis from fetal or postnatal male mice. In the present study, we examined whether functional male gametes could be obtained by the transplantation of pieces of testicular tissue from cloned mice that died immediately after birth with typical aberrant phenotypes, such as large offspring syndrome. Donor testicular tissues were retrieved from cloned mice that died postnatally and were transplanted into the testes of recipient nude mice. Two to three months after transplantation, the grafted donor testicular tissue had grown in the host testis, and histological analysis showed that spermatogenesis occurred within the graft. Intracytoplasmic sperm injection demonstrated that the testicular sperm generated in the grafted donor tissue were able to support full-term development of progeny. These results clearly showed that functional spermatogenesis could be induced by transplanting testicular tissue from cloned mice that died postnatally into recipient mice. The strategy presented here will be applicable to cloned animals of other species, because the xenografting of testicular tissue into mice has been demonstrated previously to be possible.     

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.     

Remyelination of the Adult Demyelinated Mouse Brain by Grafted Oligodendrocyte Progenitors and the Effect of B-104 Cografts

The 4e transgenic mouse is characterized by overexpression of the PLP gene. Heterozygous littermates containing three PLP gene copies develop and myelinate normally. However, a progressive CNS demyelination begins at 3-4 months of age. Despite focal demyelination, these animals survive for one year with hind limb paralysis. We used this CNS demyelination model to determine if grafts of CG4 oligodendrocyte progenitors would survive and myelinate the adult CNS. Either CG4 cells, or co-grafts of CG4/B104 cells 11:1 ratio respectively) were performed. Grafted cells survived and migrated in the normal and transgenic brain. Non-treated transgenic animals revealed extensive lack of myelin. Three months post-transplant hosts with CG4 or co-transplants displayed a near normal myelin pattern. Double immunofluorescence for neurofilament and myelin basic protein revealed the presence of many naked axons in non-grafted transgenic animals. Those grafted with progenitor CG4 cells or cografts displayed a clear increase in remyelination. This data provides a new direction for the development of cell replacement therapies in demyelinating diseases.     

Superparamagnetic iron oxide nanoparticles as a tool to track mouse neural stem cells in vivo

Cell transplantation offers a promising approach in many neurological disorders. Neural stem (NS) cells are potential candidates for cell therapy. The ability to track the grafted cells in the host tissue will refine this therapy. Superparamagnetic iron oxide nanoparticles (SPION) have been suggested as a feasible method, but there is no consensus about its safety. Here we investigated the feasibility of label NS cells with SPION and track by MRI after transplantation into mouse striatum with SPION cells and its therapeutic effects by grafting the cells into mouse striatum. We demonstrated that SPION-labeled NS cells display normal patterns of cellular processes including proliferation, migration, differentiation and neurosphere formation. Transmission electron microscopy reveals SPION in the cytoplasm of the cells, which was confirmed by microanalysis. Neurons and astrocytes generated from SPION-labeled NS cells were able to carry nanoparticles after 7 days under differentiation. SPION-labeled NS cells transplanted into striatum of mice were detected by magnetic resonance imaging (MRI) and microscopy 51 days later. In agreement with others reports, we demonstrated that NS cells are able to incorporate SPION in vitro without altering the stemness, and can survive and be tracked by MRI after they have been grafted into mice striatum.

     

A green fluorescent protein kinase substrate allowing detection and localization of intracellular ERK/MAP kinase activity

We describe a versatile intracellular reporter of ERK/MAP kinase activity: a cDNA construct, pGFP.MBP, encoding amino acids 85-144 of the human myelin basic protein fused to the C-terminus of an enhanced green fluorescent protein (GFP). The fused fragment of myelin basic protein contains a single consensus ERK/MAP kinase phosphorylation motif (PRTP, where the threonine is phosphorylated). Phosphorylation of the specific motif can be detected via immunoblotting or immunofluorescence with a commercially available phospho-specific monoclonal antibody. When expressed in mammalian cells by either transient or stable transfection, the fusion protein acts as a bona fide kinase substrate, as demonstrated by rapid serum-induced phosphorylation that is blocked by a specific MEK inhibitor. Moreover, the localization of the total substrate pool is easily visualized by GFP autofluorescence and the extent of its phosphorylation simultaneously detected within intact fixed cells by immunofluorescence using the commercially available phospho-specific antibody. The approach described should be generally applicable to the intracellular analysis of many specific protein kinase substrates for which phospho-specific antibodies have been produced.     

Successful histocompatible myoblast transplantation in dystrophin- deficient mdx mouse despite the production of antibodies against dystrophin

Myoblast transplantation has been considered a potential treatment for some muscular disorders. It has proven very successful, however, only in immunodeficient or immunosuppressed mice. In this study, myoblasts from C57BL10J +/+ mice were transplanted, with no immunosuppressive treatment, in the tibialis anterior of fully histocompatible but dystrophin-deficient C57BL10J mdx/mdx mice. One to 9 months after transplantation, the success of the graft was evaluated by immunohistochemistry. All the transplanted mice (n = 24) developed dystrophin-positive fibers following transplantation. Depending on myoblast cultures, transplantations, and time of analysis, the mice presented 15 to 80% of dystrophin-positive fibers in transplanted muscles. These fibers were correctly oriented and they were either from donor or hybrid origin. The dystrophin-positive fibers remained stable up to 9 months. Possible humoral and cellular immune responses were investigated after grafting. Antibodies directed against dystrophin and/or muscle membrane were developed by 58% of the mice as demonstrated by immunohistochemistry and Western blotting. Despite the presence of these antibodies, dystrophin-positive fibers were still present in grafted muscles 9 months after transplantation. Moreover, the muscles did not show massive infiltration by CD4 cells, CD8 cells, or macrophages, as already described in myoblast allotransplantations. This lack of rejection was attributed to the sequestrated nature of dystrophin after fiber formation. These results indicate that myoblast transplantation leads to fiber formation when immunocompetent but fully histocompatible donors and recipients are used and that dystrophin incompatibility alone is not sufficient to induce an immunological rejection reaction.     

Multimodal imaging of stem cell implantation in the central nervous system of mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s). Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting. BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin). MRI on the other hand is a non-invasive technique which is clinically applicable and can be used to precisely locate cellular grafts with very high resolution, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies. In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).     

MYELIN BASIC PROTEIN PHOSPHATASE ACTIVITY IN RAT BRAIN

Abstract— Previous work from this and other laboratories has demonstrated phosphorylation of myelin BP in vivo and in vitro. The rapid turnover of BP phosphate has suggested the presence of a phosphatase. The present studies have identified two BP phosphatases. One is present in the cytosol of rat brain homogenate. It has the highest specific activity (37 pmol/min/mg) and total activity of BP phosphatase present in any subcellular fraction. The partially purified cytosol enzyme can readily dephosphorylate soluble ³²P-labelled BP but is only half as effective in dephosphorylating membrane-bound BP. Conversely, the phosphatase which remains associated with highly purified myelin is 2.3 times as effective on BP in the membrane (7.2 pmol/min/mg) as on soluble BP (3.2 pmol/min/mg). The myelin phosphatase is tightly bound to the membrane and cannot be removed with concentrated salt solutions. During development the specific activity of the cytosol phosphatase remains constant. The specific activity of the myelin phosphatase, however, is twice as high during the period of maximum myelin formation (6.8 pmol/min/mg at 18 days) as it is in adult myelin (3.2 pmol/min/mg at 12 weeks).
In order to compare enzyme effectiveness under the various conditions employed in these studies, we have assumed that both soluble and particulate substrates are phosphorylated at equivalent sites on the polypeptide. We have further assumed that soluble and/or particulate substrates are dephosphorylated at equivalent sites on the polypeptide chain and that the various particulate and soluble enzymes have comparable access to the substrate. Within the limitations of these assumptions, our data suggest myelin phosphatase may play a significant role in phosphate turnover of BP.     

Transplantation of Foetal Neural Stem Cells into the Rat Hippocampus During Trimethyltin-Induced Neurodegeneration

The present study investigates the survival and fate of neural stem cells/progenitor cells (NSC/NPCs) homografted into the hippocampus of rats treated with trimethyltin (TMT), a potent neurotoxicant considered a useful tool to obtain a well characterized model of neurodegeneration, to evaluate their possible role in the reparative mechanisms that accompany neurodegenerative events. NSC/NPCs expressing eGFP by lentivirus-mediated infection were stereotaxically grafted into the hippocampus of TMT-treated animals and controls. Two weeks after transplantation surviving NSC/NPCs were detectable in 60% of TMT-treated animals and 30% of controls, while 30 days after transplantation only 40% of TMT-treated animals showed surviving grafted cells, which were undetectable in controls. At both times investigated, while grafted NSC/NPCs differentiated into neurons or astrocytes could be observed in addition to undifferentiated NSC/NPCs, we did not find evidence of structural integration of grafted cells into the main site of hippocampal lesion leading to appreciable repair. Maria Concetta Geloso and Stefano Giannetti contributed equally to this work.     

PARTIAL CHARACTERIZATION OF A NEW MYELIN PROTEIN COMPONENT

A new protein component has been demonstrated in myelin isolated from rat whole brain and from white matter dissected from bovine, dog and rabbit brain. It is also present in rabbit optic nerve. It does not appear to be present in other subcellular fractions of rat brain. It has a molecular weight of 20,540 ± 490(S.D.), as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. A rapid procedure for the isolation of myelin is also described.     

Islet alpha cell number is maintained in microencapsulated islet transplantation

Islet transplantation can reverse hyperglycaemia in Type 1 diabetes patients. One problem in islet transplantation is a loss of beta cell mass as well as blunted glucagon responses from the grafted islets. It has been suggested that alpha cell loss is associated with close contact of the alpha cells with the implantation organ. In the present study we made use of microencapsulation, where transplanted islets are not in direct contact with the host implantation site. After transplantation, the number of glucagon cells stained per microencapsulated islet section was increased whereas the number of insulin cells stained was decreased. DNA content of the islets was reduced, as was insulin content, whereas glucagon content was unchanged. This indicates that cell number in transplanted microencapsulated islets diminishes, which can be accounted for by loss of beta cells. However, in contrast to previous studies using non-encapsulated islets, alpha cell number seems to be maintained.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.     

Enhanced infarct myocardium repair mediated by thermosensitive copolymer hydrogel-based stem cell transplantation

Mesenchymal stem cell (MSC) transplantation by intramyocardial injection has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, low retention and survival of grafted MSCs hinder its further application. In this study, copolymer with N-isopropylacrylamide/acrylic acid/2-hydroxylethyl methacrylate-poly(ɛ-caprolactone) ratio of 88:9.6:2.4 was bioconjugated with type I collagen to construct a novel injectable thermosensitive hydrogel. The injectable and biocompatible hydrogel-mediated MSC transplantation could enhance the grafted cell survival in the myocardium, which contributed to the increased neovascularization, decreased interstitial fibrosis, and ultimately improved heart function to a significantly greater degree than regular MSC transplantation. We suggest that this novel hydrogel has the potential for future stem cell transplantation.     

The Role of Peripheral Myelin Protein 2 in Remyelination

The protein component of the myelin layer is essential for all aspects of peripheral nerves, and its deficiency can lead to structural and functional impairment. The presence of peripheral myelin protein 2 (P2, PMP2, FABP8, M-FABP) in Schwann cells has been known for decades and shown recently to be involved in the lipid homeostasis in the peripheral neural system. However, its precise role during de- and remyelination has yet to be elucidated. To this end, we assessed remyelination after sciatic nerve crush injury in vivo, and in an experimental de/remyelination ex vivo myelinating culture model in P2-deficient (P2 ^?/? ) and wild-type (WT) animals. In vivo, the nerve crush paradigm revealed temporal structural and functional changes in P2 ^?/? mice as compared to WT animals. Concomitantly, P2 ^?/? DRG cultures demonstrated the presence of shorter internodes and enlarged nodes after ex vivo de/remyelination. Together, these data indicate that P2 may play a role in remyelination of the injured peripheral nervous system, presumably by affecting the nodal and internodal configuration.     

Long-term survival of transplanted induced pluripotent stem cell-derived neurospheres with nerve conduit into sciatic nerve defects in immunosuppressed mice

Since the advent of induced pluripotent stem cells (iPSCs), clinical trials using iPSC-based cell transplantation therapy have been performed in various fields of regenerative medicine. We previously demonstrated that the transplantation of mouse iPSC-derived neurospheres containing neural stem/progenitor cells with bioabsorbable nerve conduits promoted nerve regeneration in the long term in murine sciatic nerve defect models. However, it remains unclear how long the grafted iPSC-derived neurospheres survived and worked after implantation. In this study, the long-term survival of the transplanted mouse iPSC-derived neurospheres with nerve conduits was evaluated in high-immunosuppressed or non-immunosuppressed mice using in vivo imaging for the development of iPSC-based cell therapy for peripheral nerve injury. Complete 5-mm long defects were created in the sciatic nerves of immunosuppressed and non-immunosuppressed mice and reconstructed using nerve conduits coated with iPSC-derived neurospheres labeled with ffLuc. The survival of mouse iPSC-derived neurospheres on nerve conduits was monitored using in vivo imaging. The transplanted iPSC-derived neurospheres with nerve conduits survived for 365 days after transplantation in the immunosuppressed allograft models, but only survived for at least 14 days in non-immunosuppressed allograft models. This is the first study to find the longest survival rate of stem cells with nerve conduits transplanted into the peripheral nerve defects using in vivo imaging and demonstrates the differences in graft survival rate between the immunosuppressed allograft model and immune responsive allograft model. In the future, if iPSC-derived neurospheres are successfully transplanted into peripheral nerve defects with nerve conduits using iPSC stock cells without eliciting an immune response, axonal regeneration will be induced due to the longstanding supportive effect of grafted cells on direct remyelination and/or secretion of trophic factors.     

Integration of Grafted Neural Progenitor Cells in a Host Hippocampal Circuitry after Ischemic Injury

The induction of development of neurons and glial cells from neural progenitor cells (NPCs) is at present considered a promising strategy for recoverу after ischemic insult-evoked damage to the brain. To estimate whether grafted NPCs can develop morphological properties of the mature neurons and become functionally integrated within a host hippocampal circuitry, immunohistochemical approaches at the light and electron microscopy levels have been used. Ischemic insult in FVB-strain mice was evoked by 20-min-long occlusion of both carotid arteries. One day after occlusion, NPCs from GFP-transgenic fetuses were suboccipitally transplanted into the ischemic brain. We found that 44.7 ± 3.8% (mean ± s.e.m) of the grafted GFP-positive cells differentiated 3 months after transplantation into cells demonstrating morphological features of hippocampal pyramidal neurons. Moreover, grafted cells demonstrated manifestations of rather intense formation of the synapses between host and donor neural cells. Thus, our observations show that the NPC-based transplantation approach may be promising in the treatmen t of ischemic insult.     

Two-dimensional crystal structures of protein kinase C-delta, its regulatory domain, and the enzyme complexed with myelin basic protein

Two-dimensional crystals of protein kinase C (PKC) delta, its regulatory domain (RDdelta), and the enzyme complexed with the substrate myelin basic protein have been grown on lipid monolayers composed of phosphatidylcholine: phosphatidylserine: diolein (45:50:5, molar ratio). Images have been reconstructed to 10-A resolution. The unit cells of all three proteins have cell edges a = b and interedge angle gamma = 60 degrees. RDdelta has an edge length of 33 +/- 1 A, and its reconstruction is donut shaped. The three-dimensional reconstructions from the PKCdelta C1b crystal structure () can be accommodated in this two-dimensional projection. Intact PKCdelta has an edge length of 46 +/- 1 A in the presence or absence of a nonhydrolyzable ATP analog, AMP-PnP. Its reconstruction has a similar donut shape, which can accommodate the C1b domain, but the spacing between donuts is greater than that in RDdelta; some additional structure is visible between the donuts. The complex of PKCdelta and myelin basic protein, with or without AMP-PnP, has an edge length of 43 +/- 1 A and a distinct structure. These results indicate that the C1 domains of RDdelta are tightly packed in the plane of the membrane in the two-dimensional crystals, that there is a single molecule of PKCdelta in the unit cell, and that its interaction with myelin basic protein induces a shift in conformation and/or packing of the enzyme.     

Endogenous Lectin Cerebellar Soluble Lectin Involved in Myelination Is Absent from Nonmyelinating Schwann Cells

In the sciatic nerve, two major classes of Schwann cells are present which differ in their capability to produce myelin. Myelinating Schwann cells surround most of the axons with the formation of a typical myelin sheath. Nonmyelinating Schwann cells serve to insulate individual axons without formation of myelin. These dissimilarities between the two types of Schwann cells provided an interesting model for studying mechanisms underlying myelination and the formation of contacts between axons and myelinating cells. It is demonstrated here that the endogenous lectin cerebellar soluble lectin (CSL), implicated in myelin stabilization and in formation of contact between axon and myelinating cells in the CNS and in the sciatic nerve, is undetectable in non-myelinating Schwann cells. In contrast, most axons surrounded by these cells contained the major axonal glycoprotein ligand of CSL, a 31-kDa glycoprotein which is present in large amounts. The possible relationship between the presence of CSL in Schwann cells and their capacity to interact with axons and to produce myelin are discussed.     

Amniotic membrane transplantation in ocular surface disorders

Chronic ocular surface disorders, which can result in severe functional impairment, have been viewed for decades as untreatable diseases. In 1995, the reintroduction of amniotic membrane transplantation (AMT), either alone or associated with limbal stem cell transplantation, has offered new hope of using tissue and cell therapy strategies to repair ocular surface disorders. Amniotic membrane (AM) has been found to exert its effects by acting as a substrate for the growth of ocular surface epithelia, by suppressing inflammation and scarring and by serving as an anti-microbial barrier. Moreover, AM has recently been used as a substrate for ex vivo expansion of corneal epithelial cells for ocular surface reconstruction. Notwithstanding the substantial agreement among Authors regarding its clinical efficacy, there are still many uncertainties regarding the fate of grafted AM and consequently the mechanisms through which it exerts its long-term effects. Further studies including controlled clinical trials with numerous cases are required to understand which ocular surface conditions are certain to benefit from AM transplantation and how its mechanical properties interact with the mediators produced to favor ocular surface reconstruction.     

Investigations on myelinogenesisin vitro: I. The occurrence of endogenous protein kinase and its role in the phosphorylation of myelin basic proteins in “Myelin-like membranes” isolated from cerebral cell cultures

The presence of a protein kinase capable of phosphorylating endogenous as well as exogenously added myelin basic proteins has been demonstrated in a myelin-like membrane fraction isolated from reaggregating and surface adhering, primary cultures of cells dissociated from embryonic mouse brain. Only the large and small components of myelin basic proteins were found to be phosphorylated when myelin-like membrane fraction was incubated with [-³²P]ATP. The protein kinase endogenous to the myelin-like membrane fraction was mainly of the cyclic AMP independent type. There was very little cyclic AMP dependent or cyclic GMP dependent protein kinase activities in this myelin-like fraction. Although the myelin basic proteins were the only endogenous proteins phosphorylated, protein kinase of the myelin-like membrane was capable of catalyzing the phosphorylation of exogenous substrates, such as histones.