Louisa Wirthlin

Mesenchymal stem cells for the treatment of neurodegenerative disease

Mesenchymal stem cells/marrow stromal cells (MSCs) present a promising tool for cell therapy, and are currently being tested in US FDA-approved clinical trials for myocardial infarction, stroke, meniscus injury, limb ischemia, graft-versus-host disease and autoimmune disorders. They have been extensively tested and proven effective in preclinical studies for these and many other disorders. There is currently a great deal of interest in the use of MSCs to treat neurodegenerative diseases, in particular for those that are fatal and difficult to treat, such as Huntingtons disease and amyotrophic lateral sclerosis. Proposed regenerative approaches to neurological diseases using MSCs include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation into the brain, MSCs promote endogenous neuronal growth, decrease apoptosis, reduce levels of free radicals, encourage synaptic connection from damaged neurons and regulate inflammation, primarily through paracrine actions. MSCs transplanted into the brain have been demonstrated to promote functional recovery by producing trophic factors that induce survival and regeneration of host neurons. Therapies will capitalize on the innate trophic support from MSCs or on augmented growth factor support, such as delivering brain-derived neurotrophic factor or glial-derived neurotrophic factor into the brain to support injured neurons, using genetically engineered MSCs as the delivery vehicles. Clinical trials for MSC injection into the CNS to treat traumatic brain injury and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of neurodegenerative disorders are discussed.

In Vivo Distribution of Human Adipose-Derived Mesenchymal Stem Cells in Novel Xenotransplantation Models

The potential for human adipose‐derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune‐deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human β‐globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor‐derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell‐based therapies that involve a wide range of end tissue targets.

Widespread nonhematopoietic tissue distribution by transplanted human progenitor cells with high aldehyde dehydrogenase activity

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell‐based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into β‐glucuronidase (GUSB)‐deficient NOD/SCID/mucopolysaccharidosis type VII mice, we characterized the distribution of lineage‐depleted human umbilical cord blood‐derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA‐A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true nonhematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of nonhematopoietic cells. However, relying solely on continued expression of cell surface markers, as used in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH‐expressing progenitor cells demonstrated widespread and tissue‐specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage.

Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease

Bone marrow‐derived mesenchymal stem cells (MSCs) are a promising platform for cell‐ and gene‐based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD‐SCID MPSVII]). Primary human bone marrow‐derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme β‐glucuronidase (MSCs‐GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD‐SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs‐GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs‐GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.

In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

Differentially Expressed MicroRNAs in Chondrocytes from Distinct Regions of Developing Human Cartilage

There is compelling in vivo evidence from reports on human genetic mutations and transgenic mice that some microRNAs (miRNAs) play an important functional role in regulating skeletal development and growth. A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation (i.e. production of progenitor, differentiated or hypertrophic chondrocytes) is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54–56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair.

Molecular properties and fibril ultrastructure of types II and XI collagens in cartilage of mice expressing exclusively the α1(IIA) collagen isoform

Until now, no biological tools have been available to determine if a cross-linked collagen fibrillar network derived entirely from type IIA procollagen isoforms, can form in the extracellular matrix (ECM) of cartilage. Recently, homozygous knock-in transgenic mice (Col2a1(+ex2), ki/ki) were generated that exclusively express the IIA procollagen isoform during post-natal development while type IIB procollagen, normally present in the ECM of wild type mice, is absent. The difference between these Col2a1 isoforms is the inclusion (IIA) or exclusion (IIB) of exon 2 that is alternatively spliced in a developmentally regulated manner. Specifically, chondroprogenitor cells synthesize predominantly IIA mRNA isoforms while differentiated chondrocytes produce mainly IIB mRNA isoforms. Recent characterization of the Col2a1(+ex2) mice has surprisingly shown that disruption of alternative splicing does not affect overt cartilage formation. In the present study, biochemical analyses showed that type IIA collagen extracted from ki/ki mouse rib cartilage can form homopolymers that are stabilized predominantly by hydroxylysyl pyridinoline (HP) cross-links at levels that differed from wild type rib cartilage. The findings indicate that mature type II collagen derived exclusively from type IIA procollagen molecules can form hetero-fibrils with type XI collagen and contribute to cartilage structure and function. Heteropolymers with type XI collagen also formed. Electron microscopy revealed mainly thin type IIA collagen fibrils in ki/ki mouse rib cartilage. Immunoprecipitation and mass spectrometry of purified type XI collagen revealed a heterotrimeric molecular composition of α1(XI)α2(XI)α1(IIA) chains where the α1(IIA) chain is the IIA form of the α3(XI) chain. Since the N-propeptide of type XI collagen regulates type II collagen fibril diameter in cartilage, the retention of the exon 2-encoded IIA globular domain would structurally alter the N-propeptide of type XI collagen. This structural change may subsequently affect the regulatory function of type XI collagen resulting in the collagen fibril and cross-linking differences observed in this study.

Human stem cells for tissue repair.

The primary application for hematopoietic stem cell transplantation has traditionally been to reconstitute blood cell lineages that had formed abnormally because of genetic mutations, or that had been eradicated to treat a disease such as leukemia. However, in recent years, compelling data has suggested that stem cells from the bone marrow might have far greater potential, and might be able to repair damaged or diseased tissues outside of the blood-forming compartment. Much attention has been paid to the concept of ‘‘stem cell plasticity,’’ and the hope that stem cells could be used to repair damaged tissues has generated immense excitement in many fields.

Human Hematopoietic Cell Culture, Transduction, and Analyses

This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34+ cells, transduction of these cells with a retroviral vector on fibronectin‐coated plates, assaying the efficiency of transduction, and establishing long‐term cultures. Support protocols describe methods for maintenance of vector‐producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long‐term cultures. Other protocols provide PCR‐based methods to analyze individual colonies for transduction, methods to analyze cells harvested from long‐term cultures, and procedures for freezing and thawing of hematopoietic cells. Curr. Protoc. Hum. Genet. 56:13.7.1‐13.7.31.

460. Transplantation of Human Aldehyde Dehydrogenase Expressing Cells Leads to Widespread Tissue Distribution of Donor Cells in the Pancreas and Liver of NOD/SCID/MPSVII Mice

Beta-glucuronidase (GUSB) is a lysosomal enzyme expressed in virtually all cell types. Transplantation of GUSB-expressing human hematopoietic stem cells (HSC) into the GUSB-deficient NOD/SCID/MPSVII mouse allows for the detection of xenotransplanted cells in hematopoietic and non-hematopoietic tissues, without reliance on the expression of human-specific cell surface markers or in situ hybridization (Hoffing, 2003). We are currently using the NOD/SCID/MPSVII model to investigate the transfer of the GUSB gene from human cells into murine cells of GUSB deficient tissues. We recently characterized a novel population of reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin-) and selection of cells with high aldehyde dehydrogenase (ALDH) activity (Hess, 2004). These ALDHhiLin- cells demonstrate robust hematopoietic engraftment in the NOD/SCID model and contain a subpopulation of cells (ALDHhiCD133+) that may not be fully restricted to hematopoietic lineages. Here, we have used the NOD/SCID/MPSVII model to study the tissue distribution of ALDHhiLin- cells in multiple organs. Tail vein injection of 2|[times]|105 ALDHhiLin- cells into sub-lethally irradiated (300cGy) NOD/SCID/MPSVII mice (n=6) demonstrated hematopoietic chimerism in the BM (70.5|[plusmn]|15.1%), spleen (7.0|[plusmn]|2.8%) and peripheral blood (17.0|[plusmn]|10.7%). In contrast, injection of 106 ALDHloLin- cells (n=5) did not produce consistent human cell engraftment. By using GUSB-specific histochemical staining, significant human engraftment was also detected in the non-hematopoietic (liver, pancreas, kidney, lung, heart, and brain) tissues of mice transplanted with ALDHhiLin- cells. GUSB activity was co-localized with CD45 expression in the BM of mice transplanted with ALDHhiLin- cells. However, flow cytometric analysis of liver tissue revealed a discrepancy between engraftment detected by a fluorescent GUSB substrate, compared to human CD45 cell surface expression. Histochemical staining confirmed the presence of GUSB+ cells that did not express human CD45, and uncovered GUSB expressing cells with typical hepatocyte morphology. These hepatocyte-like GUSB+ cells were also present after the transplantation of the ALDHhiCD133+Lin- subpopulation (n=4). In the pancreas, GUSB+ donor cells were localized in ductal regions and surrounding recipient islets. The ALDHhiLin- UCB cells demonstrated a previously uncharacterized high level of engraftment in non-hematopoietic organs of NOD/SCID MPSVII mice. We are currently investigating cellular fusion and nuclear reprogramming as a putative mechanism for the production of non-hematopoietic cells expressing donor cell-derived GUSB. This gene transfer potentially occurs through spontaneous fusion as previously demonstrated in liver (Willenbring, 2004) and muscle (Camargo, 2004). This phenomenon may be further exploited to deliver target genes into various tissues for the treatment of genetic abnormalities.