Changes in type II procollagen isoform expression during chondrogenesis by disruption of an alternative 5' splice site within Col2a1 exon 2.

This study describes a new mechanism controlling the production of alternatively spliced isoforms of type II procollagen (Col2a1) in vivo. During chondrogenesis, precursor chondrocytes predominantly produce isoforms containing alternatively spliced exon 2 (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. We previously identified an additional Col2a1 isoform containing a truncated exon 2 and premature termination codons in exon 6 (type IIC). This transcript is produced by utilization of another 5' splice site present in exon 2. To determine the role of this IIC splicing event in vivo, we generated transgenic mice containing silent knock-in mutations at the IIC 5' splice site (Col2a1-mIIC), thereby inhibiting production of IIC transcripts. Heterozygous and homozygous knock-in mice were viable and display no overt skeletal phenotype to date. However, RNA expression profiles revealed that chondrocytes in cartilage from an age range of Col2a1-mIIC mice produced higher levels of IIA and IID mRNAs and decreased levels of IIB mRNAs throughout pre-natal and post-natal development, when compared to chondrocytes from littermate control mice. Immunofluorescence analyses showed a clear increase in expression of embryonic type II collagen protein isoforms (i.e. containing the exon 2-encoded cysteine-rich (CR) protein domain) in cartilage extracellular matrix (ECM). Interestingly, at P14, P28 and P56, expression of embryonic Col2a1 isoforms in Col2a1-mIIC mice persisted in the pericellular domain of the ECM in articular and growth plate cartilage. We also show that persistent expression of the exon 2-encoded CR domain in the ECM of post-natal cartilage tissue may be due, in part, to the embryonic form of type XI collagen (the α3 chain of which is also encoded by the Col2a1 gene). In conclusion, expression of the Col2a1 IIC splice form may have a regulatory function in controlling alternative splicing of exon 2 to generate defined proportions of IIA, IID and IIB procollagen isoforms during cartilage development. Future studies will involve ultrastructural and biomechanical analysis of the collagen ECM to determine the effects of persistent mis-expression of embryonic collagen isoforms in mature cartilage tissue.

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.

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.

Quantification of type II procollagen splice forms using alternative transcript-qPCR (AT-qPCR).

During skeletal development, the onset of chondrogenic differentiation is marked by expression of the α1(II) procollagen (Col2a1) gene. Exon 2 of Col2a1 codes for a cysteine-rich von Willebrand factor C-like domain. Chondroprogenitors express the exon 2-containing IIA and IID splice forms by utilizing adjacent 5' splice sites separated by 3 base pairs. There is a shift to expression of the shorter, exon 2-lacking IIB splice form with further differentiation. Alternative splicing analysis of Col2a1 splice forms has often relied upon semi-quantitative PCR, using a single set of PCR primers to amplify multiple splice forms. We show that this widely used method is inaccurate due to mismatched amplification efficiency of different-sized PCR products. We have developed the TaqMan®-based AT-qPCR (Alternative Transcript-qPCR) assay to more accurately quantify alternatively spliced mRNA, and demonstrate the measurement of Col2a1 splice form expression in differentiating ATDC5 cells in vitro, and in wild type mouse embryonic and postnatal cartilage in vivo. The AT-qPCR assay is based on the use of a multiple-amplicon standard (MAS) plasmid, containing a chemically synthesized cluster of splice site-spanning PCR amplicons, to quantify alternative splice forms by standard curve-based qPCR. The MAS plasmid designed for Col2a1 also contained an 18S rRNA amplicon for sample normalization, and an amplicon corresponding to a region spanning exon 52-53 to measure total Col2a1 mRNA. In mouse E12.5 to P70 cartilages, we observed the expected switch between the IIA and IIB splice forms; no IID or IIC splice products were observed. However, in the ATDC5 cultures, predominant expression of the IIA and IID splice forms was found at all times in culture. Additionally, we observed that the sum of the IIA, IIB and IID splice forms comprises only a small fraction of Col2a1 transcripts containing the constitutive exon 52-53 junction. We conclude from our results that the majority of ATDC5 cells in the assay described in this study remained as chondroprogenitors during culture in standard differentiation conditions, and that additional Col2a1 transcripts may be present. The validity of this novel AT-qPCR assay was confirmed by demonstrating the expected Col2a1 isoform expression patterns in vivo in developing mouse cartilage. The ability to measure true levels of procollagen type II splice forms will provide better monitoring of chondrocyte differentiation in other model systems. In addition, the AT-qPCR assay described here could be applied to any gene of interest to detect and quantify known and predicted alternative splice forms and can be scaled up for high throughput assays.

Disruption of the developmentally-regulated Col2a1 pre-mRNA alternative splicing switch in a transgenic knock-in mouse model.

The present study describes the generation of a knock-in mouse model to address the role of type II procollagen (Col2a1) alternative splicing in skeletal development and maintenance. Alternative splicing of Col2a1 precursor mRNA is a developmentally-regulated event that only occurs in chondrogenic tissue. Normally, chondroprogenitor cells synthesize predominantly exon 2-containing mRNA isoforms (type IIA and IID) while Col2a1 mRNA devoid of exon 2 (type IIB) is the major isoform produced by differentiated chondrocytes. Another isoform, IIC, has also been identified that contains a truncated exon 2 and is not translated into protein. The biological significance of this IIA/IID to IIB splicing switch is not known. Utilizing a splice site targeting knock-in approach, a 4 nucleotide mutation was created to convert the 5' splice site of Col2a1 exon 2 from a weak, non-consensus sequence to a strong, consensus splice site. This resulted in apparent expression of only the IIA mRNA isoform, as confirmed in vitro by splicing of a type II procollagen mini-gene containing the 5' splice site mutation. To test the splice site targeting approach in vivo, homozygote mice engineered to retain IIA exon 2 (Col2a1(+ex2)) were generated. Chondrocytes from hindlimb epiphyseal cartilage of homozygote mice were shown to express only IIA mRNA and protein at all pre- and post-natal developmental stages analyzed (E12.5, E16.5, P0, P3, P7, P14, P28 and P70). As expected, type IIB procollagen was the major isoform produced in wild type cartilage at all post-natal time points. Col2a1(+ex2) homozygote mice are viable, appear healthy and display no overt phenotype to date. However, research is currently underway to investigate the biological consequence of persistent expression of the exon 2-encoded conserved cysteine-rich domain in post-natal skeletal tissues.

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 Huntington's 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.

Contribution of human hematopoietic stem cells to liver repair.

Immune-deficient mouse models of liver damage allow examination of human stem cell migration to sites of damage and subsequent contribution to repair and survival. In our studies, in the absence of a selective advantage, transplanted human stem cells from adult sources did not robustly become hepatocytes, although some level of fusion or hepatic differentiation was documented. However, injected stem cells did home to the injured liver tissue and release paracrine factors that hastened endogenous repair and enhanced survival. There were significantly higher levels of survival in mice with a toxic liver insult that had been transplanted with human stem cells but not in those transplanted with committed progenitors. Transplantation of autologous adult stem cells without conditioning is a relatively safe therapy. Adult stem cells are known to secrete bioactive factors that suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate recruitment, retention, mitosis, and differentiation of tissue-residing stem cells. These paracrine effects are distinct from the direct differentiation of stem cells to repair tissue. In patients at high risk while waiting for a liver transplant, autologous stem cell therapy could be considered, as it could delay the decline in liver function.

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.

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 beta-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.

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.

Immunodeficient mouse models to study human stem cell-mediated tissue repair.

Hematopoietic stem cell transplantation has traditionally been used 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, much attention has been paid to the new concept of "stem cell plasticity," and the hope that stem cells could be used to repair damaged tissues generated immense excitement. The field is now in a more realistic and critical period of intense investigation and the concept of cell fusion to explain some of the observed effects has been shown after specific types of damage in liver and muscle, both organs that contain a high number of multinucleate cells. The field is still an extremely exciting one, and many questions remain to be answered before stem cell therapy for tissue repair can be used effectively in the clinic. Immune deficient mouse models of tissue damage provide a system in which human stem cell migration to sites of damage and subsequent contribution to repair can be carefully evaluated. This chapter gives detailed instructions for methods to study human stem cell contribution to damaged liver and to promote repair of damaged vasculature in immune deficient mouse models.

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 beta-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. ALDH(hi) or ALDH(hi)CD133+ 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.

Human progenitor cells rapidly mobilized by AMD3100 repopulate NOD/SCID mice with increased frequency in comparison to cells from the same donor mobilized by granulocyte colony stimulating factor.

AMD3100 inhibits the interaction between SDF-1 and CXCR4, and rapidly mobilizes hematopoietic progenitors for clinical transplantation. However, the repopulating function of human cells mobilized with AMD3100 has not been characterized in comparison to cells mobilized with granulocyte-colony stimulating factor (G-CSF) in the same donor. Therefore, healthy donors were leukapheresed 4 hours after injection with AMD3100; after 10 days of drug clearance the same donor was mobilized with G-CSF, allowing a paired comparison of repopulation by mobilized cells. Transplantation of mononuclear cells (MNC) or purified CD34(+) cells was compared at limiting dilution into NOD/SCID mice. Human AMD3100-mobilized MNC possessed enhanced repopulating frequency in comparison to G-CSF-mobilized MNC from paired donors, and purified CD34(+) progenitors were at least as efficient as the G-CSF mobilized cells. The frequencies of NOD/SCID repopulating cells (SRC) were 1 SRC in 8.7 x 10(6) AMD3100-mobilized MNC compared to 1 SRC in 29.0 x 10(6) G-CSF-mobilized MNC, and 1 SRC in 1.2 x 10(5) AMD3100-mobilized CD34(+) cells compared to 1 SRC in 1.8 x 10(5) G-CSF-mobilized CD34(+) cells. Hematopoietic differentiation of transplanted progenitors was similar after AMD3100 or G-CSF-mobilization. Thus, AMD3100 mobilized peripheral blood represents a rapidly obtained, highly repopulating source of hematopoietic progenitors for clinical transplantation.

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 beta-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.

Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells.

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH(hi)Lin- cells). Here, we further dissected the ALDH(hi)-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDH(hi)CD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDH(hi)CD133-Lin- and ALDH(hi)CD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDH(hi)CD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID beta2M-null mice that received transplants of ALDH(hi)CD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDH(hi)CD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies.

Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity.

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture, purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population, compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse, suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype.

Glial cell line-derived neurotrophic factor-enriched bridging transplants promote propriospinal axonal regeneration and enhance myelination after spinal cord injury.

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-beta (TGF-beta) family, is widely expressed in the developing and adult central nervous system (CNS). At present, limited information is available regarding the effects of GDNF in the repair of spinal cord injury (SCI). In the present study, mini-guidance channels containing either: (1) Matrigel (MG, a basement membrane component), (2) Schwann cells (SCs, 120 x 10(6)/ml) in MG (SC-MG), (3) recombinant human GDNF (rhGDNF, 3 microg/microl) in MG (GDNF-MG), and (4) a combination of all three components (GDNF-SC-MG) were grafted into a T9 hemisection-gap lesion in adult rats to examine the effects of GDNF on axonal regeneration and myelination following SCI. Thirty days post-transplantation, limited axonal growth was observed within guidance channels containing MG-alone (MG). When SCs were added to the channels (SC-MG group), consistent axonal ingrowth containing both myelinated and unmyelinated axons was observed, confirming our previous findings. The addition of GDNF-alone without SCs (GDNF-MG) resulted in substantial ingrowth of unmyelinated axons, suggesting that GDNF has a direct neurite-growth promoting effect on these axons. Implantation of channels containing both GDNF and SCs (GDNF-SC-MG) produced a significant and synergistic increase in axonal regeneration and myelination. In addition, GDNF reduced the extent of reactive gliosis, infiltration of activated macrophages/microglia, and cystic cavitation at the graft-host interfaces. Retrograde tracing revealed that grafts of SC-seeded channels containing GDNF promoted a significant increase in the number of propriospinal neurons which had regenerated their axons into the grafts, as compared to SC-MG-seeded channels. These results indicate that GDNF may play a novel therapeutic role in promoting propriospinal axonal regeneration, enhancing myelin formation, and improving graft-host interfaces after SCI.