Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.researchgate.net/publication/10632674 Transplantationofporcineumbilicalcord Matrixcellsintotheratbrain,Exp ArticleinExperimentalNeurology·September2003 ImpactFactor:4.7·DOI:10.1016/S0014-4886(03)00128-6·Source:PubMed CITATIONS READS 112 14 7authors,including: MarkLouisWeiss JulieEHix 43PUBLICATIONS1,311CITATIONS KansasStateUniversity SEEPROFILE 1PUBLICATION112CITATIONS SEEPROFILE DerylTroyer KansasStateUniversity 135PUBLICATIONS3,437CITATIONS SEEPROFILE Allin-textreferencesunderlinedinbluearelinkedtopublicationsonResearchGate, lettingyouaccessandreadthemimmediately. Availablefrom:DerylTroyer Retrievedon:09May2016 Available online at www.sciencedirect.com R Experimental Neurology 182 (2003) 288 –299 www.elsevier.com/locate/yexnr Transplantation of porcine umbilical cord matrix cells into the rat brain M.L. Weiss,a,* K.E. Mitchell,a J.E. Hix,a S. Medicetty,a S.Z. El-Zarkouny,b D. Grieger,b and D.L. Troyera a Department of Anatomy and Physiology, Kansas State University, College of Veterinary Medicine, Manhattan, KS 66506-5602, USA b Department of Animal Science and Industry, Kansas State University, Manhattan, KS 66506-5602, USA Received 1 August 2002; revised 28 October 2002; accepted 4 February 2003 Abstract Immune rejection of transplanted material is a potential complication of organ donation. In response to tissue transplantation, immune rejection has two components: a host defense directed against the grafted tissue and an immune response from the grafted tissue against the host (graft vs host disease). To treat immune rejection, transplant recipients are typically put on immunosuppression therapy. Complications may arise from immune suppression or from secondary effects of immunosuppression drugs. Our preliminary work indicated that stem cells may be xenotransplanted without immunosuppression therapy. Here, we investigated the survival of pig stem cells derived from umbilical cord mucous connective tissue (UCM) after transplantation into rats. Our data demonstrate that UCM cells survive at least 6 weeks without immune suppression of the host animals after transplantation into either the brain or the periphery. In the first experiment, UCM cells were transplanted into the rat brain and recovered in that tissue 2– 6 weeks posttransplantation. At 4 weeks posttransplantation, the UCM cells engrafted into the brain along the injection tract. The cells were small and roughly spherical. The transplanted cells were positively immunostained using a pig-specific antibody for neuronal filament 70 (NF70). In contrast, 6 weeks posttransplantation, about 10% of the UCM cells that were recovered had migrated away from the injection site into the region just ventral to the corpus callosum; these cells also stained positively for NF70. In our second experiment, UCM cells that were engineered to constitutively express enhanced green fluorescent protein (eGFP) were transplanted. These cells were recovered 2– 4 weeks after brain transplantation. Engrafted cells expressing eGFP and positively staining for NF70 were recovered. This finding indicates a potential for gene therapy. In the third experiment, to determine whether depositing the graft into the brain protected UCM cells from immune detection/clearance, UCM cells were injected into the tail vein and/or the semitendinosis muscle in a group of animals. UCM cells were recovered from the muscle or within the kidney 3 weeks posttransplantation. In control experiments, rat brains were injected with PKH 26-labeled UCM cells that had been lysed by repeated sonic disruption. One and 2 weeks following injection, no PKH 26-labeled neurons or glia were observed. Taken together, these data indicate that UCM cells can survive xenotransplantation and that a subset of the UCM cells respond to local signals to differentiate along a neural lineage. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Stem cells; Umbilical cord mesenchyme; Neural differentiation; Graft vs host disease Introduction Dr. Gerald D. Fischbach put it nicely: “Stem cells that can develop into a variety of different types of nerve cells and glia would be extremely valuable in the therapy of acute and chronic neurological disorders.” (Larsen, 1999). In the * Corresponding author. Department of Anatomy and Physiology, Kansas State University, Coles Hall 105, Manhattan, KS 66506-5602, USA. Fax: ⫹1-785-532-4557. E-mail address: [email protected] (M.L. Weiss). CNS, neural stem cell populations have been identified as ependymal cells (Johansson et al., 1999), subventricular zone astrocytes (Doetsch et al., 1999), and cells in the subgranular zone of the dentate gyrus (Ray et al., 1993). Thus, harvesting neural stem cells from the human patient cannot be easily done because the location and distribution of the neural stem cells makes it difficult to harvest a substantial population of these cells for in vitro expansion/ manipulation and subsequent autologous transplantation. Therefore, a question remains: Where can we get the neural progenitor cells for transplantation? 0014-4886/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4886(03)00128-6 M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 Two potential sources of neural stem cells have been identified: first, the “transdifferentiation” of adult stem cells derived from other primordial lineages, such as those derived from adult bone marrow stromal (BMS) cells, adipose tissue, and hematopoietic tissue. BMS cells, for example, are pluripotent cells that can differentiate into bone, cartilage, fat, muscle, tendon, neurons, and many other tissues (Pittenger et al., 1999; Prockop, 1997). There is evidence that BMS cells transplanted into rats can “home” in on pathology. For example, BMS cells transplanted into rats with induced liver damage contribute to the formation of new hepatic oval cells that further differentiate into hepatocytes and ductal epithelium (Peterson et al., 1999). BMS cells home in on damaged muscle in irradiated mice as well (Ferrari et al., 1998; Gussoni et al., 1999). Kopen et al. (1999) showed that BMS cells injected intracerebroventricularly (ICV) migrate extensively and differentiate into glial cells and cells that express neurofilament (a neuronspecific marker) in neonatal mice. Two labs reported that after bone marrow hematopoietic cells were injected intravenously, they migrated and expressed neuronal and microglial antigens in CNS (Brazelton et al., 2000; Mezey et al., 2000). In vitro adult rat and human BMS cells can be induced to differentiate into neurons (Woodbury et al., 2000). A potential drawback of using BMS cells for autologous transplantation may be the difficulty in obtaining sufficient material from older, diseased patients to expand and differentiate. A second source of stem cells is from embryonic stem (ES) cell lines. ES cells are pluripotent and immortal (Bain et al., 1995; Brustle et al., 1999; Deacon et al., 1998; Okabe et al., 1996). ES cells have been successfully induced to express neural phenotypes (Lee et al., 2000; Spenger et al., 1994, 1995; Studer et al., 1995, 1996; Yan et al., 2001). Several potential problems exist with transplanting ES cells: First, in a significant percentage of cases, ES cells form teratomas when transplanted into rodent brain (Thomson et al., 1998). Second, to avoid rejection of grafted material, donor recipients are typically put on immunosupression therapy. Serious complications can arise from immune suppression and from the secondary effects of the immunosuppressive drugs, such as cyclosporine A (Kaplan, 1998; Sander et al., 1996). Third, a dilemma exists about the moral cost/societal benefit of therapeutic use of human ES cells. Here, we present a potential source of stem cells for neural transplantation: cells derived from umbilical cord mesenchyme (UCM), also known as Wharton’s jelly mesenchyme or mucous connective tissue. In a preliminary report (Mitchell et al., 2003), we demonstrated that pig UCM cells are stem cells that can be differentiated into neurons and glia, can be maintained and grown indefinitely in vitro, and can be genetically manipulated to express exogenous genes. Here, pig UCM cells were found in the brains of rats following transplantation without immunosuppressive therapy and a population of the UCM cells appear to differentiate into neurons and migrate from the injection site. 289 Methods Stem cell culture The culture method is elaborated in another article (Mitchell et al., 2003). Briefly, pig umbilical cords were aseptically collected from preterm fetuses (approximately 60-day) at slaughter. Umbilical arteries and vein were stripped manually and discarded. The remaining tissue was minced finely in a sterile container in DMEM media with an antibiotic (Gentamycin, 20 g/ml, GIBCO BRL) and an antifungal agent (Amphotericin B 250 g/l, Sigma). The explants were transferred to six-well plates containing the above media along with 20% fetal bovine serum (FBS) for culture. The primary cultures were left undisturbed for about 7 days to allow migration of cells from the explants, and then refed. They were fed thereafter twice weekly and passaged as necessary (cells passaged at 80 –90% confluency). These stem cell cultures have been maintained beyond 100 population doublings and continue to grow vigorously (see Fig. 1). Enhanced green fluorescent protein (eGFP)-expressing UCM cells The UCM cells were modified by S.Z. El-Zarkouny and D. Grieger. The Sleeping Beauty Transposon system (a generous gift from P. Hackett) was modified as follows: The plasmid containing the transposon pT/HygR-eGFP (also a gift from Hackett lab) was used as the template to generate a PCR product of the hygromycin resistance-eGFP insert. The neomycin resistance gene from the original transposon vector (pT/SVNeo) was removed using the blunt cutters BsaB1 and Nac1, and the hygromycin R/eGFP PCR product was ligated into the original vector. We cotransfected this plasmid along with the pCMV-SB plasmid containing the transposase gene driven by the CMV promoter using lipofection (Lipofectamine, BRL). Hygromycin was added to the medium after 3 days at 200 or 250 g/ml to select for transfected cells and stable transfection was attained after 3 weeks in selection media. The eGFP-expressing UCM cells were maintained in hygromycin containing medium for two to three passages prior to transplantation. Transplantation procedure UCM cells that had been in culture for 17, 40, 57, 58, 60 passages were used for the transplantation experiments. There were no apparent differences in the results that could be attributed to using one passage or another. In some cases, the UCM cells were labeled with the lipophilic dye PKH 26 red (Sigma, St. Louis, MO) prior to transplantation. PKH 26 is a nontoxic permanent fluorescent marker (Ashley et al., 1993; Honig and Hume, 1989). To transplant, the preconfluent cells were lifted with trypsin (7– 8 min). The trypsin was inactivated by the addition of an equal volume of 290 M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 Fig. 1. Phase contrast micrograph of uninduced UCM cells in culture. Two cell types are obvious: flat, fibroblast-like layer that adheres to the substrate with scattered small round cells (arrows). When the cells become confluent, rounded clusters of cells that float above the substrate start appearing (dotted circle). The culture can be sustained by passaging either the clusters or the adherent cells without apparent differences. DMEM and 20% fetal bovine serum. The cells from several plates were pooled and the number of cells was estimated by counting on a hemocytometer. The final concentration was adjusted to about 1000 cells/l. The UCM cells were transplanted into anesthetized Lewis or Sprague–Dawley rats (2% halothane in oxygen) centrally via stereotaxic injection (approximately 10,000 cells in 10 l) or into the periphery via tail vein injection (approx. 106 cells in 0.5 ml flushed with 0.5 ml sterile saline) and intramuscular injection (approx. 106 cells in 0.4 ml), or via intramuscular injection alone (approx. 106 cells in 0.5 ml). For stereotaxic injection, a glass micropipette was lowered into the striatum (Bregma ⫹0.5, Lateral 3.4; D-V 5.0) and a 1-l bolus of graft cells delivered over 1 min. After a 1-min interval, the micropipette was raised approximately 200 m and a second 1-l injection made. In this way, multiple injections were distributed along an injection tract until the entire 10-l volume was delivered. In other cases, the animals had a guide cannula implanted in a previous surgical session prior to delivering the cells via an injection cannula (Plastics One). Control transplants (sterile saline alone, Con rats) were performed in age-matched animals. In a separate control experiment, two rats were transplanted with PKH 26-labeled UCM cells that had been previously lysed by sonic disruption in phosphate buffer saline (for approximately 1 min). The disruption of the cells was confirmed by flow cytometry after exposure to sonic disruption and by plating an aliquot of the disrupted cells in growth media. Control rats and normal animals with no treatment served as specificity and background controls for immunocytochemistry. All animal manipulations were conducted in accordance with PHS and Society for Neuroscience guidelines and with the prior approval of the IACUC. Immunocytochemistry Previously described methods for detection of a single antigen were used (Fitch et al., 2000; Fitch and Weiss, 2000). Briefly, Equithesin-anesthetized rats were sacrificed by transcardial perfusion with heparinized isotonic saline rinse followed by 10% buffered neutral formalin. The brains were removed, postfixed 2 h, and cryoprotected in 20% sucrose. Frozen sections were cut at 30 – 40 m coronally and sections were collected into three sets of adjacent sections, one set consisting of every third serial section. One set of sections was processed for IC and the adjacent sets were held in reserve in a cryoprotectant solution (Watson et al., 1986). Free-floating tissue sections were immunocytochemically stained for GFP (Chemicon), pig-specific neurofilament 70 (NF70) (Chemicon), neuron-specific -tubulin (TuJ1) (Covalence Research Products), NFM (Chemicon), and 2⬘, 3⬘-cyclic nucleotide-3⬘-phosphodiesterase (CNPase) (Chemicon) and localized either with immunofluorescence or with peroxidase using a commercially available ABC kit (VectaStain). The monoclonal NF70 antibody was particularly valuable because it does not recognize rodent neurofilaments. When using this antibody, it was necessary to substitute previously adsorbed secondary antibody (adsorbed for rat antigens, Jackson Labs) for the secondary provided in the Vectastain kit. The tissue was incubated in the following reagents: endogenous peroxidase elimination, 5% blocking serum, followed by the primary anti-serum, fluorophore-labeled (Jackson Immuno, fluoroscein isothiocyanate or Molecular Probes, Alexafluor 480) or biotinlabeled (from the VectaStain kit) secondary antibody. The tissue was triple rinsed with PBS– 0.2% Triton X-100 between each incubation. For immunofluorescence detection, M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 the tissue were mounted on gelatin-chromium potassium sulfate-coated microscope slides and air-dried. The stained sections were examined using epifluorescence microscopy after clearing and coverslipping with glycerol containing N-propyl gallate to prevent fading. For immunoperoxidase detection, the antigen was localized with diaminobenzidine (DAB, Sigma) and hydrogen peroxide. Immunocytochemical (IC) labeling was considered positive if the signal was distinctly above background and the signal was above that seen in the negative controls (omission of primary antibody, or labeling found in control or normal rats). To be considered double-labeled, the morphology and location of the cell must appear identical in both brightfield (DAB) and fluorescence (PKH 26 or eGFP). Results Pig UCM cells in culture Details about the cell culture, propagation and maintenance of pig UCM cells can be found in Mitchell et al., (2003). Briefly, when UCM cells initially grow outward from explants two morphologically distinct populations of cells are present: spherical or flat mesenchymal cells. When the cells become confluent, they form spherical colonies that remain attached to cells below (see Fig. 1). These colonies resemble “neurospheres” (Reynolds and Weiss, 1992). UCM cell culture can be maintained either by harvesting the neurosphere-like cell clusters or by passage of preconfluent flat and spherical cells without apparent differences. We have maintained UCM cell cultures for more than 100 population doublings and they continue to grow vigorously. Three cellular characteristics indicate that undifferentiated UCM cells are a type of stem cell: (1) the number of passages that they have been maintained in culture; (2) the fact that UCM cells are telomerase-positive; and (3) UCM cells make the receptor for stem cell factor, c-kit (data found in Mitchell et al., 2003). UCM cells have been characterized in vitro by immunocytochemistry and Western blotting (Mitchell et al., 2003). UCM cells can be induced to differentiate into neurons and glia following the procedure described by Woodbury et al. (2000) (see Fig. 2, further details in Mitchell et al., 2003). A small percentage of untreated UCM cells and a larger percentage of differentiated UCM cells exhibit positive staining for neural proteins. Within 1 h of induction treatment, multiple “neurites” can be seen extending from many cells, and the cell bodies become rounded and refractile in phase contrast (Mitchell et al., 2003). Fig. 2 shows how UCM cells respond to induction using the Woodbury et al. protocol and examples of differentiated UCM cells that are IC positive for TuJ1, tau, and NFM (see Mitchell et al., 2003, for full details). Here, we transplanted undifferentiated, preconfluent pig UCM cells into adult rats. 291 Experiment 1. Transplantation of pig UCM cells into rat brain Two injection methods were used with differing results. The Hamilton syringe delivery method produced much less tissue damage and a more discrete graft compared to the animals that received the guide cannula implantation and subsequent grafting via a secondary injection cannula. In addition, the injection through the injection cannula was more damaging to the UCM cells than the microliter syringe (data not shown). In the animals injected with the Hamilton syringe, the graft cells were located along the injection tract and no gross brain damage was found 4 weeks after injection despite the large volume (4 l) and many UCM cells were found along the injection tract (see Fig. 3). In contrast, the guide cannula animals had more extensive damage to the brain associated with the larger diameter guide cannula (data not shown). Apparently the brain tissue adjacent to the implanted cannula had withdrawn slightly because the transplanted cells were found distributed adjacent to the guide cannula tract, as well as at the tip. Following recovery from surgery, no complications were observed. No animals died subsequent to transplantation and no unusual behaviors were noted. There was no sign of brain tumor or teratoma, immunological response, or glioma in transplant recipients; all animals increased body weight. Four weeks after transplantation, there was no apparent glial scar. Large injection volumes and guide cannula implantation caused obvious tissue damage, as one would expect. Multiple nuclei, indicative of fusion with host cells, were not observed in the transplanted cells and there was no evidence of uncontrolled replication of UCM cells after transplantation. After tissue processing, pig UCM cells were identified either by PKH 26 fluorescent staining of dye-loaded cells or by pig-specific NF70 immunocytochemical staining (see Fig. 3–5). PKH 26 fluorescent staining was found throughout the cytoplasm and membrane. NF70 immunocytochemical staining was spread throughout the cell cytoplasm (see Fig. 3–5). No NF70 or PKH 26 staining was found in control animals. There was no evidence of immunological response in the 2- to 8-week period after grafting; i.e., there was no perivascular cuffing, no extracellular debris, and no phagocytosis. A subset of the UCM cells had migrated into the parenchyma of the brain away from the injection site. At 2– 4 weeks posttransplantation, most UCM cells appeared as simple spherical cells 10 –15 m in diameter with a granulated cytoplasm. A small subset of the UCM cells had single short processes extending from the cell body at this time. Posttransplantation, many UCM cells were found along the injection tract (see Fig. 3). At 6 weeks, UCM cells were also found ipsilateral to the transplantation site adjacent to the corpus callosum. Thus, a subset of UCM cells had apparently migrated from the injection site into the parenchyma (data not shown). At 2– 6 weeks posttransplantation, a subset of the PKH 26-labeled UCM cells were immunocytochemically stained 292 M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 Fig. 2. UCM cells induced to a neural phenotype in culture. (A–C) Change in cell morphology is shown in sequential photographs of a culture that was induced to differentiate along the neural lineage (see text). (A) Uninduced UCM cells. (B) Same culture of cells after exposure to the full-term induction (FI) protocol. (C) Same cells after 10 days in the long-term induction media (LTI). (FI and LTI protocols described in detail in Woodbury et al. (2000). Inset: Note long processes and phase-bright cell body. The neurite-like short processes (arrows) and the growth cone-like projection at the distal end of a long process. (D–F) Differentiated UCM cells demonstrate positive IC staining for neural markers: class III neuron-specific -tubulin (TuJ1) (D), neurofilament medium (NF-M) (E), or a neuron-specific microtubule-associated protein, tau (F). (G) High-power phase-contrast micrograph of cell exposed to LTI protocol. Note: The granular material that resembles Nissl substance and the “neurites” with primary and secondary processes. M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 293 Fig. 3. UCMs 2 weeks after transplantation. The same field is shown on the top and bottom. Left panels: Transplanted cells were identified by the PKH 26 dye loading (top) or by the expression of pig-specific NF70 immunocytochemical staining (bottom). The enclosed area is shown at higher magnification on the right. Right panels: The top panel shows the relatively simple morphology of the UCMs 2 weeks after transplantation into the rat brain. For the most part, the cells lack processes, have a granular cytoplasm, and stain brightly with the PKH 26 dye. The bottom panel shows immunocytochemical staining for pig-specific NF70. The arrows indicate examples of cells that are both PKH 26-stained and positively immunocytochemically stained for NF70. The circles indicate UCMs cells that do not stain for NF70, suggesting that not all UCMs differentiate along the neural lineage following transplantation. The asterisks indicate cells that positively immunocytochemically stain for NF70 (bottom), but do not stain with PKH 26. The interpretation of this result is that the PKH 26 dye loading did not stain 100% of the UCMs. for neural markers such as TuJ1 and MAP2 (see Fig. 4). Positive staining for CNPase in some PKH 26 cells was also detected, suggesting that some of the UCM cells may differentiate into oligodendrocytes (data not shown). It was interesting to note that TuJ1, CNPase, and MAP2 staining was found in PKH 26-negative cells that may not be part of the grafted material (indicated by asterisks in Fig. 4). Experiment 2. Transplantation of eGFP-expressing UCM cells into the brain At 4 weeks posttransplantation, eGFP-expressing UCM cells were detected in the brain spread along the cannula tract. The cytoplasm of these cells had a granular appearance and a large percentage of the cells stained positively 294 M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 Fig. 4. Transplanted UCMs express neural-specific markers in rat brain. (A) Left: Pig UCMs, indicated by PKH 26 staining, 4 weeks after transplantation into rat brain. Note that the PKH 26 staining is usually confined to the cell bodies. Right: Identical field as shown in the left panel. TuJ1 immunocytochemical staining. UCMs that stain for the neural-specific marker TuJ1 are indicated by arrows. Arrowheads indicate PKH-labeled fibers that stain positively for TuJ1. (A) Asterisks indicate PKH 26-positive cells that do not stain for TuJ1. This may indicate that not all graft cells differentiate along the neural lineage. (B) Left: Pig UCMs 4 weeks after transplantation. Right: IC staining for the -III tubulin protein (a neuronal marker), TuJ1. The filled circles indicate the large number of double-labeled cells. (B) Asterisks indicate TuJ1-stained cells in the graft that may not originate from the graft (lack PKH 26 staining). This would suggest that the graft may stimulate endogenous stem cell migration and differentiation. The arrowheads indicate the location of TuJ1 IC-positive fibers. (C) M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 295 Fig. 5. Pig UCM cells were injected into the periphery. In this case, the UCM cells were delivered intramuscularly into the semitendinosis and intravenously. Left: PHK 26-labeled cells are found along the im injection tract 4 weeks after injection. Right: PKH 26-labeled cells were found within the parenchyma of the kidney. Finding transplanted UCM cells 4 weeks after injection suggests that the immune system does not clear these cells from the body. for NF70 (see Fig. 4). The graft cells can be identified by eGFP fluorescence or by using an anti-GFP antibody 2– 8 weeks posttransplantation (data not shown). With either detection method, GFP staining found throughout the cell cytoplasm and thus the morphology of the grafted cells was revealed. The morphology of GFP-stained cells was simple spherical or fusiform with zero, one, or two processes. The exogenous nature of the eGFP-expressing cells was confirmed by double staining for pig-specific NF70 (see Fig. 4). Extracellular GFP staining was never observed. There was no evidence of phagocytosis or extracellular debris in the 2to 8-week survival period. In control animals, no eGFP staining was observed around the injection site. living cells were found in vitro. An aliquot containing 10,000 lysed cells was injected into two rats. One rat survived 1 week; the other survived 2 weeks after injection prior to sacrifice and tissue processing. In both cases, cellular debris or red blood cells were found along the injection tract (see Fig. 6B). No fluorescent labeling was found within neurons or glia. On occasion, fluorescent blood cells were observed along the injection tract (see arrows in Fig. 6B); the red blood cells were easily distinguished from the PKH 26-labeled UCM cells by their smaller size and smooth, round, or doughnut-like appearance. Discussion Experiment 3. Peripheral injection of pig UCM cells UCM cells were injected into the periphery, intramuscularly (N ⫽ 3) or both intramuscularly (IM) and intravenously (IV) (N ⫽ 1), in a group of rats. Three weeks after IM injection, PKH 26-labeled UCM cells were recovered from the injection site (see Fig. 5). Three weeks after IM and IV injection, PKH 26-labeled UCM cells were engrafted in the parenchyma of the kidney (see Fig. 6). No IC characterization was performed in these cases. Experiment 4. Brain injection of disrupted pig UMC cells Flow cytometry indicated that sonic disruption fragmented the cells prior to transplantation (see Fig. 6A). No Here, we present four lines of evidence indicating that pig UCM cells are stem cells that do not stimulate immune rejection when transplanted into the adult rat. First, pig UCM cells survive 2– 6 weeks after transplantation into the rat without immune suppression therapy. Second, pig UCM cells respond to differentiation cues and modify their morphology and neurochemical phenotype to resemble neural cells both in cell culture and after transplantation into the rat brain. At 2 and 4 weeks after injection, most pig UCM cells found in the rat brain were simple spherical cells with a granular cytoplasm. At 6 weeks, some UCM cells had migrated from the injection site to a site adjacent to the corpus callosum and had short processes. Third, pig UCM cells that were injected into the periphery were recovered in Same field is shown on the left and right. Left panel: engrafted UCM cells. Right panel: IC staining for neuron-specific microtubule-associated protein 2 (MAP2). The filled circles indicate the double-labeled cells. The asterisks indicate MAP2-stained cells that may not be of graft origin (they lack PKH 26). The arrowheads indicate MAP2 IC-positive fibers. (D) Left: UCM cells that were engineered to express eGFP were detected 4 weeks after transplantation. Note that most of the cells have a granular cytoplasm and a few have short primary processes. Right: Many of the eGFP-expressing cells also stain for pig-specific NF70, confirming that they are from porcine origin. The filled circles indicate corresponding areas in both fields. There was a large percentage of double-labeled cells. 296 M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 Fig. 6. Previously disrupted PKH26-labeled UCM cells do not label neurons or glia in rat brain following transplantation. (A) Flow cytometry was used to assess the fragmentation of pig UCM cells following sonic disruption. Left: UCM cells prior to disruption (Control). Middle: Flow cytometry data following sonic disruption indicates that 0.3% of the population remains within the gated region. Right: Following a second round of sonic disruption, 0.2% of the population remains within the gated region. Culturing of an aliquot of the lysate did not yield cells. Taken together, these data indicate that the UCM cells were destroyed prior to transplantation. (B) Top: One week after injection of disrupted PKH26-labeled cells, the area along the injection track was examined. While the background fluorescence was higher along the injection track, red blood cells were the only fluorescent cells found in this area (indicated by the arrowheads). No fluorescent neurons or glia were observed. Bottom: In contrast, when intact PKH 26-labeled UCM cells are injected, fluorescent cells were recovered in and around the injection tract 2– 6 weeks following injection (data from a 4-week survival postinjection is shown here). Note that the fluorescent graft cells (indicated by triangles in bottom panel) are larger and more irregular in appearance than the small, doughnut-shaped red blood cells indicated in the top panel. Calibration bar ⫽ 20 m. the injection site and the kidney 3 weeks later. Fourth, after injection of disrupted UCM cells, no cells labeled by PKH 26 were found. Together these results indicate that pig UCM cells are relatively nonimmunogenic, that they respond to local cues found in the adult rat, and that these cells engraft without stimulating significant immune rejection. M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 The properties of UCM cells in culture While umbilical cord blood banking has become a reality with identified clinical significance (Huhn, 2000; Jacobs and Falkenburg, 1998; Nishihira et al., 1998; Rendine et al., 2000; Wagner and Kurtzberg, 1998), the loose connective tissue matrix of the umbilical cord, e.g., the Wharton’s Jelly matrix, is not widely appreciated as a source of stem cells. Eyden et al. and McElreavey et al. recognized myofibroblast-like cells in the umbilical cord matrix (Eyden et al., 1994; McElreavey et al., 1991), but apparently that work was not pursued. We speculated and subsequently demonstrated that the umbilical cord Wharton’s Jelly is a rich source of primitive mesenchymal stem cells (Mitchell et al., 2003). Pig UCM cells appear poised to differentiate into neurons and are rapidly induced along this pathway and express neural markers such as TuJ1, NF-M, and tau in vitro (for further characterization, see Mitchell et al., 2003). Thus, we were interested to determine the fate of pig UCM cells after transplantation into adult rat brain. It is interesting to speculate that stem cells derived from UCM may serve as the source of the persistent fetal cells found in the mother (Bianchi, 1996). It is possible that some special property of the UCM stem cells may permit them to evade the host immune system. Here, pig UCM cells were isolated from Wharton’s jelly, the matrix of the umbilical cord, rather than cord blood. Because umbilical cord blood has been found to be a source of hematopoietic stem cells (Broxmeyer et al., 1989, 1990; Gluckman, 1996; Lu and Ende, 1997), we confirmed that the UCM cells used here are not derived from cord blood. Umbilical blood vessels were stripped from the cord before explant preparation, and the UCM cells tested negative for markers of the hematopoietic lineage (such as CD34 and CD45; Weiss et al., unpublished observations). Transplantation and recovery of UCM cells After tissue processing, the transplanted cells were identified in three different ways. First, the UCM cells that were loaded with PKH 26 prior to transplantation were recovered by observing fluorescent cells, and not fluorescent debris, along the injection track and elsewhere in the brain. Red blood cells (RBCs) also fluoresce and were found in the brains of transplanted and control animals but RBCs were easily differentiated from the transplanted UCM cells based upon their size and shape (see Fig. 6). Injection of disrupted PKH 26-labeled UCM cells did not label host neurons or glia. This indicates that the lysed PKH 26-labeled UCM cells do not stain host cells following phagocytosis, possibly due to enzymatic degradation of the dye. Second, many, but not all, transplanted UCM cells were identified by their staining for the pig-specific NF70 IC staining 2– 6 weeks after introduction. The NF70 antibody does not recognize rodent epitopes, and in these experiments NF70 IC staining was never found in normal or control animals. In contrast, 297 NF70 staining was often colocalized with PKH 26 fluorescence. Most important, NF70 staining was not found in debris, phagocytic cells, or lysozomal vesicles. Third, the UCM cells that were engineered to produce eGFP prior to transplantation were recovered either by observing eGFP using epifluorescence or by IC staining for the GFP protein and immunoperoxidase. IC staining for GFP would not be expected in the case of UCM cell lysis because the released GFP protein and mRNA is likely to be degraded by phagocytic cells. In both cases, eGFP was found in transplanted animals, but not in either control group. All three of these recovery methods indicated that the transplanted cells were found in the rat brain 2– 6 weeks after injection. Further, our results indicated that the transplanted cells do not form tumors. We cannot at this time address whether the UCM cells replicate after transplantation. Ongoing work involving the transplantation of 150 eGFP-expressing UCM cells and a time series analysis indicates that UCM cells may replicate between transplantation and a 2- to 8-week recovery period (Medicetty et al., in preparation). While we did not directly evaluate the infiltration of lymphocytes, macrophages, natural killer cells, microglia, or astrooytes, our findings suggest that the grafted cells were not recognized or attacked. There was no indication of cellular lysis or cellular debris in animals transplanted with UCM cells and there were no obvious signs of immunological response, such as infiltration of immune cells, perivascular cuffing, extracellular debris, or graft antigens within cells with glial morphology or size. In contrast, when lysed cells were injected into the brain, debris and RBCs were found in the injection site. Further, because UCM cells were recovered in the kidney 3 weeks after peripheral injection or up to 6 weeks after injection into the brain, we believe that UCM cells avoid immune surveillance. These findings are in contrast to previous findings by Larsson et al., who transplanted fetal pig ventral mesencephalic tissue (E27 or E29) into rat brain and demonstrated immune rejection in 2– 4 weeks (Larsson et al., 2000). There are several possible explanations for these disparate results. One possible explanation is that primitive undifferentiated stem cells do not have the full complement of surface antigens, and thus do not stimulate immune rejection. This explanation is in agreement with recent work performed on human ES cells which indicated that the expression of MHC class I proteins correlated with the differentiation of ES cells (Drukker et al., 2002). A second explanation may be that extraembryonic tissues, such as the umbilical cord, have some endogenous factors that suppress immune recognition, and stem cells derived from this tissue may be immunosuppressive, as discussed above. Previous work had indicated a significant reduction in the immune response to expanded neural precursor cells compared with primary fetal tissue suspensions (Armstrong et al., 2001). In Armstrong et al.’s study, small but significant levels of porcine major histocompatibility complex (MHC) expression were found in the primary tissue suspensions and no expression was found in expanded 298 M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 neural precursor cells. From the present results, it is unknown whether MHC expression or lack thereof accounts for the survival of the transplanted material. limiting factor for successful grafting. Pig UCM cells appear to overcome this limitation and, thus, may prove to be therapeutically useful in the future. Fate of transplanted tissue Acknowledgments Implanted UCM cells primarily developed into neural grafts as indicated by IC staining for pig-specific NF70 at 2– 6 weeks after transplantation and by the IC staining for other neuron-specific markers weeks after transplantation. For example, positive double-labeling of UCM cells for two other cytoskeletal markers also identified the transplanted cells as neurons: class III TuJ1, and microtubule-associated protein 2. In contrast, fewer oligodendrocytes originated from the grafted material, as indicated by the few cells that double-stained for CNPase and PKH 26. It was noted that associated with the grafted tissue, pig-specific NF70-stained cells were found that did not contain PHK 26 (these cells are indicated by asterisks in Fig. 3 and 4). We interpret this result as indicating that not all of the UCM cells were labeled in vitro prior to transplantation. The observed immunocytochemical staining of UCM cells shown here extends our previous studies of in vitro cells that had indicated that UCM cells could be induced to differentiate into neurons and glia (see Fig. 2 and Mitchell et al., 2003). Therapeutic potential of UCM cells In Parkinson’s disease (PD), human fetal mesencephalic transplants were once used as the source of replacement cells, but moral/ethical concerns associated with the use of fetal tissue and the scarcity of the tissue and other problems associated with obtaining enough tissues have been barriers to the widespread use of transplantation. Currently, ventral mesencephalic cells obtained from fetal pigs are used for xenotransplants in PD patients (Bjorklund and Lindvall, 2000; Deacon et al., 1997). Xenotransplants have problems such as graft vs host disease or immune rejection (Larsson et al., 2000). For therapeutically useful numbers of the xenografted cells to survive, the host’s immune system must be suppressed (Deacon et al., 1997). Despite immune suppression, it was estimated that about 5–10% (Bjorklund, 1991) or about 25% (Brundin et al., 2000). of the harvested cells survive transplantation. We have begun to analyze human UCM stem cells and found that they can be induced to differentiate along the neural lineage in vitro (Medicetty, et al., unpublished observations), just as we have shown for pig UCM cells (see Fig. 2 and Mitchell et al., 2003). Currently, we are investigating whether human UCM cells lack cell surface antigens (MHC class I and II proteins) and whether the expression of those proteins change after differentiation along the neural lineage (Drukker et al., 2002). This work, along with efforts to introduce clinically useful genes (Mannes et al., 1998), is important to determining the therapeutic potential of UCM cells. At present, overcoming or reducing the immune responses of the host is a critical Cameron Fahrenholtz, Lois Morales, Tammi Hildreth, and Katrina Fox are thanked for technical assistance. Dr. Duane Davis is thanked for reviewing an earlier draft of the manuscript and providing ideas and moral support. This work was supported, in part, by a KSU USRG award and by NIH NS 34160 to M.L.W.; by NIH COBRE RR15563 to K.E.M.; and by Project 481326 (DT) from the Kansas Agricultural Experiment Station. References Armstrong, R.J., Harrower, T.P., Hurelbrink, C.B., McLaughin, M., Ratcliffe, E.L., Tyers, P., Richards, A., Dunnett, S.B., Rosser, A.E., Barker, R.A., 2001. Porcine neural xenografts in the immunocompetent rat: immune response following grafting of expanded neural precursor cells. Neuroscience 106, 201–216. Ashley, D.M., Bol, S.J., Waugh, C., Kannourakis, G., 1993. A novel approach to the measurement of different in vitro leukaemic cell growth parameters: the use of PKH GL fluorescent probes. Leuk. Res. 17, 873– 882. Bain, G., Kitchens, D., Yao, M., Huettner, J.E., Gottlieb, D.I., 1995. Embryonic stem cells express neuronal properties in vitro. Dev. Biol. 168, 342–357. Bianchi, D.W., Zickwolf, G.K., Weil, G.J., Sylvester, S., Demaria, M.A., 1996. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc. Natl. Acad. Sci. USA 93, 705–708. Bjorklund, A., 1991. Neural transplantation—an experimental tool with clinical possibilities. Trends Neurosci. 14, 319 –322. Bjorklund, A., Lindvall, O., 2000. Cell replacement therapies for central nervous system disorders. Nat. Neurosci. 3, 537–544. Brazelton, T.R., Rossi, F.M., Keshet, G.I., Blau, H.M., 2000. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290, 1775–1779. Broxmeyer, H.E., Douglas, G.W., Hangoc, G., Cooper, S., Bard, J., English, D., Arny, M., Thomas, L., Boyse, E.A., 1989. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/ progenitor cells. Proc. Natl. Acad. Sci. USA 86, 3828 –3832. Broxmeyer, H.E., Gluckman, E., Auerbach, A., Douglas, G.W., Friedman, H., Cooper, S., Hangoc, G., Kurtzberg, J., Bard, J., Boyse, E.A., 1990. Human umbilical cord blood: a clinically useful source of transplantable hematopoietic stem/progenitor cells. Int. J. Cell Cloning 8 (Suppl 1), 76 – 89. Brundin, P., Karlsson, J., Emgard, M., Schierle, G.S., Hansson, O., Petersen, A., Castilho, R.F., 2000. Improving the survival of grafted dopaminergic neurons: a review over current approaches. Cell Transplant. 9, 179 –195. Brustle, O., Jones, K.N., Learish, R.D., Karram, K., Choudhary, K., Wiest ler, O.D., Duncan, I.D., Mckay, R.D., 1999. Embryonic stem cellderived glial precursors: a source of myelinating transplants. Science 285, 754 –756. Deacon, T., Dinsmore, J., Costantini, L.C., Ratliff, J., Isacson, O., 1998. Blastula-stage stem cells can differentiate into dopaminergic and serotonergic neurons after transplantation. Exp. Neurol. 149, 28 – 41. Deacon, T., Schumacher, J., Dinsmore, J., Thomas, C., Palmer, P., Kott, S., Edge, A., Penney, D., Kassissieh, S., Dempsey, P., Isacson, O., 1997. M.L. Weiss et al. / Experimental Neurology 182 (2003) 288 –299 Histological evidence of fetal pig neural cell survival after transplantation into a patient with Parkinson’s disease. Nat. Med. 3, 350 –353. Doetsch, F., Caille, I., Lim, D.A., Garcia-verdugo, J.M., Alvarez-buylla, A., 1999. Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97, 703–716. Drukker, M., Urbach, A., Schuldiner, M., Markel, G., Itskovitz-Eldor, J., Reubinoff, B., Mandelboim, O., Benvenisty, N., 2002. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc. Natl. Acad. Sci. USA 99, 9864 –9869. Eyden, B.P., Ponting, J., Davies, H., Bartley, C., Torgersen, E., 1994. Defining the myofibroblast: normal tissues, with special reference to the stromal cells of Wharton’s jelly in human umbilical cord. J. Submicrosc. Cytol. Pathol. 26, 347–355. Ferrari, G., Cusella-de angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A., Cossu, G., Mavilio, F., 1998. Muscle regeneration by bone marrowderived myogenic progenitors. Science 279, 1528 –1530. Fitch, G.K., Patel, K.P., Weiss, M.L., 2000. Activation of renal afferent pathways following furosemide treatment: I. Effects of survival time and renal denervation. Brain Res. 861, 363–376. Fitch, G.K., Weiss, M.L., 2000. Activation of renal afferent pathways following furosemide treatment: II. Effect of angiotensin blockade. Brain Res. 861, 377–389. Gluckman, E., 1996. Umbilical cord blood transplant in human. Bone Marrow Transplant. 18 (Suppl 2), 166 –170. Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A., Khan, M.K., Flint, A.F., Kunkel, L.M., Mulligan, R.C., 1999. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 401, 390 –394. Honig, M.G., Hume, R.I., 1989. Di I and Di O: versitile fluorescent dyes for neuronal labeling and pathway tracing. Trends Neurosci. 12, 333– 341. Huhn, R.D., 2000. Umbilical cord blood stem cell transplantation and banking. N. J. Med. 97, 53–57. Jacobs, H.C., Falkenburg, J.H., 1998. Umbilical cord blood banking in The Netherlands. Bone Marrow Transplant. 22 (Suppl 1), S8 –10. Johansson, C.B., Momma, S., Clarke, D.L., Risling, M., Lendahl, U., Frisen, J., 1999. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96, 25–34. Kaplan, N.M., 1998. Clinical hypertension, Williams & Wilkins, Baltimore. Kopen, G.C., Prockop, D.J., Phinney, D.G., 1999. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc. Natl. Acad. Sci. USA 96, 10711–10716. Larsen, N., Transplanted neural stem cells migrate throughout the abnormal brain, reduce disease symptoms http://www.ninds.nih.gov/news _and_events/pressrelease_neural_stem_cells_060799.htm. 6-9-1999. Larsson, L.C., Czech, K.A., Brundin, P., Widner, H., 2000. Intrastriatal ventral mesencephalic xenografts of porcine tissue in rats: immune responses and functional effects. Cell Transplant. 9, 261–272. Lee, S.H., Lumelsky, N., Studer, L., Auerbach, J.M., McKay, R.D., 2000. Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat. Biotechnol. 18, 675– 679. Lu, S., Ende, N., 1997. Potential for clinical use of viable pluripotent progenitor cells in blood bank stored human umbilical cord blood. Life Sci. 61, 1113–1123. Mannes, A.J., Caudle, R.M., O’connell, B.C., Iadarola, M.J., 1998. Adenoviral gene transfer to spinal-cord neurons: intrathecal vs. intraparenchymal administration. Brain Res. 793, 1– 6. McElreavey, K.D., Irvine, A.I., Ennis, K.T., McLean, W.H., 1991. Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton’s jelly portion of human umbilical cord. Biochem. Soc. Trans. 19, 29S. Mezey, E., Chandross, K.J., Harta, G., Maki, R.A., Mckercher, S.R., 2000. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290, 1779 –1782. 299 Mitchell, K.E., Weiss, M.L., Mitchell, B.M., Martin, P., Davis, D., Morales, L.M., Helwig, B., Beerenstrauch, M., Abou-easa, K., Medicetty, S., Hildreth, T., Troyer, D.L., 2003. Wharton’s Jelly mesenchymal cells form neurons and glia. Stem Cells 21, 50 – 60. Nishihira, H., Ohnuma, K., Ikuta, K., Isoyama, K., Kinoshita, A., Toyoda, Y., Ohira, M., Okamura, J., Nakajima, F., 1998. Unrelated umbilical cord-blood stem cell transplantation: a report from Kanagawa Cord Blood Bank, Japan. Int. J. Hematol. 68, 193–202. Okabe, S., Forsberg, N.K., Spiro, A.C., Segal, M., McKay, R.D., 1996. Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech. Dev. 59, 89 –102. Peterson, B.E., Bowden, W.C., Patrene, K.D., Mars, W.M., Sullivan, A.K., Murase, S.S., Boggs, J.S., Greenberger, J.S., Goff, J.P., 1999. Bone marrow as a potential source of hepatic oval cells. Science 284, 1168 – 1170. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–147. Prockop, D., 1997. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science 276, 71–74. Ray, J., Peterson, D.A., Schinstine, M., Gage, F.H., 1993. Proliferation, differentiation, and long-term culture of primary hippocampal neurons. Proc. Natl. Acad. Sci. USA 90, 3602–3606. Rendine, S., Curtoni, E.S., D celle, P.F., Berrino, M., Bertola, L., Barbanti, M., Saracco, P., Fazio, L., Gay, E., Dall’omo, A.M., 2000. Analysis of the Turin umbilical cord blood bank registry. Transfusion 40, 813– 816. Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710 [see comments]. Sander, M., Lyson, T., Thomas, G.D., Victor, R.G., 1996. Sympathetic neural mechanisms of cyclosporine-induced hypertension. Am. J. Hypertens. 9, 121S–138S. Spenger, C., Hyman, C., Studer, L., Egli, M., Evtouchenko, L., Jackson, C., Dahl-Jorgensen, A., Lindsay, R.M., Seiler, R.W., 1995. Effects of BDNF on dopaminergic, serotonergic, and GABAergic neurons in cultures of human fetal ventral mesencephalon. Exp. Neurol. 133, 50 – 63. Spenger, C., Studer, L., Evtouchenko, L., Egli, M., Burgunder, J.M., Markwalder, R., Seiler, R.W., 1994. Long-term survival of dopaminergic neurones in free-floating roller tube cultures of human fetal ventral mesencephalon. J. Neurosci. Methods 54, 63–73. Studer, L., Spenger, C., Seiler, R.W., Altar, C.A., Lindsay, R.M., Hyman, C., 1995. Comparison of the effects of the neurotrophins on the morphological structure of dopaminergic neurons in cultures of rat substantia nigra. Eur. J. Neurosci. 7, 223–233. Studer, L., Spenger, C., Seiler, R.W., Othberg, A., Lindvall, O., Odin, P., 1996. Effects of brain-derived neurotrophic factor on neuronal structure of dopaminergic neurons in dissociated cultures of human fetal mesencephalon. Exp. Brain Res. 108, 328 –336. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147. Wagner, J.E., Kurtzberg, J., 1998. Banking and transplantation of unrelated donor umbilical cord blood: status of the National Heart, Lung, and Blood Institute-sponsored trial. Transfusion 38, 807– 809. Watson-Re, J., Wiegand, S.J., Clough, R.W., Hoffman, G.E., 1986. Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology. Peptides 7, 155–159 [published erratum appears in Peptides 1986 May–Jun; 7(3):545]. Woodbury, D., Schwarz, E.J., Prockop, D.J., Black, I.B., 2000. Adult rat and human bone marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364 –370. Yan, J., Studer, L., Mckay, R.D., 2001. Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors. J. Neurochem. 76, 307–311.