Erratum: iPS-derived MSCs from an expandable bank to deliver a prodrug-converting enzyme that limits growth and metastases of human breast cancers.

[This corrects the article DOI: 10.1038/cddiscovery.2016.64.].

iPS-derived MSCs from an expandable bank to deliver a prodrug-converting enzyme that limits growth and metastases of human breast cancers.

One attractive strategy to treat cancers is to deliver an exogenous enzyme that will convert a non-toxic compound to a highly toxic derivative. The strategy was tested with viral vectors but was disappointing because the efficiency of transduction into tumor cells was too low. Recent reports demonstrated that the limitation can be addressed by using tissue-derived mesenchymal stromal cells (MSCs) to deliver enzyme/prodrug systems that kill adjacent cancer cells through bystander effects. Here we addressed the limitation that tissue-derived MSCs vary in their properties and are difficult to generate in the large numbers needed for clinical applications. We prepared a Feeder Stock of MSCs from induced pluripotent stem cells (iPSs) that provided an extensively expandable source of standardized cells. We then transduced the iPS-derived MSCs to express cytosine deaminase and injected them locally into a mouse xenogeneic model of human breast cancer. After administration of the prodrug (5-fluorocytosine), the transduced iPS-MSCs both limited growth of preformed tumors and decreased lung metastases.

Intra-articular injection of human mesenchymal stem cells (MSCs) promote rat meniscal regeneration by being activated to express Indian hedgehog that enhances expression of type II collagen.

OBJECTIVE: Meniscal regeneration was previously shown to be enhanced by injection of mesenchymal stem/stromal cells (MSCs) but the mode of action of the MSCs was not established. The aim of this study was to define how injection of MSCs enhances meniscal regeneration. DESIGN: A hemi-meniscectomy model in rats was used. Rat-MSCs (rMSCs) or human-MSCs (hMSCs) were injected into the right knee joint after the surgery, and PBS was injected into the left. The groups were compared macroscopically and histologically at 2, 4, and 8 weeks. The changes in transcription in both human and rat genes were assayed by species-specific microarrays and real-time RT-PCRs. RESULTS: Although the number of hMSCs decreased with time, hMSCs enhanced meniscal regeneration in a manner similar to rMSCs. hMSCs injection increased expression of rat type II collagen (rat-Col II), and inhibited osteoarthritis progression. The small fraction of hMSCs was activated to express high levels of a series of genes including Indian hedgehog (Ihh), parathyroid hormone-like hormone (PTHLH), and bone morphogenetic protein 2 (BMP2). The presence of hMSCs triggered the subsequent expression of rat-Col II. An antagonist of hedgehog signaling inhibited the expression of rat-Col II and an agonist increased expression of rat-Col II in the absence of hMSCs. CONCLUSIONS: Despite rapid reduction in cell numbers, intra-articular injected hMSCs were activated to express Ihh, PTHLH, and BMP2 and contributed to meniscal regeneration. The hedgehog signaling was essential in enhancing the expression of rat-Col II, but several other factors provided by the hMSCs probably contributed to the repair.

Nonviral gene delivery of erythropoietin by mesenchymal stromal cells.

Erythropoietin (EPO) acts on erythroblasts in the bone marrow (BM) to stimulate the formation of red blood cells. In this study, we wanted to determine whether BM-derived mesenchymal stromal cells (MSCs) can be used as cellular vehicles to deliver EPO in mice without the use of viral vectors. After isolation and characterization of murine MSCs (mMSCs), different transient transfection procedures were compared for their efficacy of gene transfer of the pEGFP-N2 plasmid. Nucleofection outperformed magnetofection and lipofection. Stably transfected mMSCs were generated by selection with G418-disulfate and single-cell-colony-forming unit (sc-CFU) assays without changes in their proliferation capacity and osteogenic/adipogenic differentiation potential. Next, murine EPO was stably introduced into mMSCs by nucleofection of a plasmid encoding the epo and egfp genes. Intraperitoneal transplantation of EPO-expressing mMSCs increased serum EPO levels, hematocrit and hemoglobin of C57BL/6 mice within 1 week. The hematocrit remained elevated for 5 weeks, but production of antibodies against both transgenes was detected in the hosts and serum EPO levels normalized. Our results suggest that nonviral gene delivery into MSCs can be used to enhance the known beneficial effects MSCs by additional production of therapeutic factors like EPO in vivo.

A potential role for Dkk-1 in the pathogenesis of osteosarcoma predicts novel diagnostic and treatment strategies.

Canonical Wnt signalling is an osteoinductive signal that promotes bone repair through acceleration of osteogenic differentiation by progenitors. Dkk-1 is a secreted inhibitor of canonical Wnt signalling and thus inhibits osteogenesis. To examine a potential osteoinhibitory role of Dkk-1 in osteosarcoma (OS), we measured serum Dkk-1 in paediatric patients with OS (median age, 13.4 years) and found it to be significantly elevated. We also found that Dkk-1 was maximally expressed by the OS cells at the tumour periphery and in vitro, Dkk-1 and RANKL are coexpressed by rapidly proliferating OS cells. Both Dkk-1 and conditioned media from OS cells reduce osteogenesis by human mesenchymal cells and by immunodepletion of Dkk-1, or by adding a GSK3beta inhibitor, the effects of Dkk-1 were attenuated. In mice, we found that the expression of Dkk-1 from implanted tumours was similar to the human tumour biopsies in that human Dkk-1 was present in the serum of recipient animals. These data demonstrate that systemic levels of Dkk-1 are elevated in OS. Furthermore, the expression of Dkk-1 by the OS cells at the periphery of the tumour probably contributes to its expansion by inhibiting repair of the surrounding bone. These data demonstrate that Dkk-1 may serve as a prognostic or diagnostic marker for evaluation of OS and furthermore, immunodepletion of Dkk-1 or administration of GSK3beta inhibitors could represent an adjunct therapy for this disease.

"Stemness" does not explain the repair of many tissues by mesenchymal stem/multipotent stromal cells (MSCs).

There has recently been an explosion of interest in adult stem/progenitor cells that have the potential to repair tissues, with over 3,000 citations to publications (PubMed) and numerous announcements of clinical trials in which the cells are used to treat individuals with a broad range of diseases. At the same time, the data present a paradox-the cells originally attracted attention because of their stem-cell-like properties, but the cells frequently repair injured tissues without much evidence of either engraftment or differentiation.

Abnormal response to physical activity in femurs after heterozygous inactivation of one allele of the Col2a1 gene for type II collagen in mice.

The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6-TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC). The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and maturation.

Correction of a mineralization defect by overexpression of a wild-type cDNA for COL1A1 in marrow stromal cells (MSCs) from a patient with osteogenesis imperfecta: a strategy for rescuing mutations that produce dominant-negative protein defects.

Gene therapy for dominant-negative disorders presents a more difficult challenge than gene therapy for recessive disorders, since even partial replacement of a protein for a recessive disorder can reverse symptoms. Osteogenesis imperfecta (OI) has frequently served as a model disorder for dominant-negative defects of structural proteins. The disease is caused by mutations in type I collagen (COL1A1), the major structural component of bone, skin and other connective tissues. The severity of the phenotype is largely dependent on the ratio of normal to mutant type I procollagen synthesized by cells. Recently, attempts have been made to develop strategies for cell and gene therapies using the adult stem cells from bone marrow referred to as mesenchymal stem cells or marrow stromal cells (MSCs). In this study, we used MSCs from a patient with type III OI who was heterozygous for an IVS 41A+4C mutation in COL1A1. A hybrid genomic / cDNA construct of COL1A1 was transfected into the MSCs and the transfectants were expanded over a 200-fold. Transfected MSCs showed increased expression of the wild-type mRNA and protein. In vitro assays demonstrated that the transfected cells more efficiently differentiated into mineralizing cells. The results indicated that it is possible to overexpress COL1A1 cDNA in OI MSCs and thereby to correct partially the dominant-negative protein defect.

Stable transfection of MSCs by electroporation.

Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 micros in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neo(r)). After electroporation of 10(6) cells with 10 microg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 microg/ml G418. The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.

Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.

STUDY DESIGN: Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen ("heterozygous knockout") were studied. OBJECTIVE: To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. SUMMARY OF BACKGROUND DATA: Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. METHODS: Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens alpha1(I) and alpha1(II). Voluntary running activity also was monitored in half of the mice. RESULTS: As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. CONCLUSION: Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.

Targeted disruption of Col11a2 produces a mild cartilage phenotype in transgenic mice: comparison with the human disorder otospondylomegaepiphyseal dysplasia (OSMED).

Transgenic mice were prepared by homologous recombination with a Col11a2 targeting gene in which an inverted neomycin-resistant gene was inserted between restriction sites in exons 27 and 28. The targeted allele was transcribed in shortened mRNAs, which could be detected by Northern blotting. However, translation of the full-length Col11a2 chain was unable to occur because of the presence of premature termination codons within the inverted neomycin-resistant gene. Analysis of pepsin-resistant collagen chains from rib cartilage of homozygous mice demonstrated the lack of synthesis of intact alpha2(XI) chains. However, pepsin-resistant collagen chains of either alpha1(XI) or alpha1(V) were still detected on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Therefore, alpha2(XI) chains are not essential for the assembly of some molecular forms of triple-helical type V/XI collagen. The phenotype was milder than in the cho/cho mouse in which, as the result of mutation, translation of the full-length alpha1(XI) chain fails to occur and the mice die at birth (Li et al., 1995). Homozygous mice without expression of an alpha2(XI) chain had a smaller body size, receding snouts, and deafness. Nasal bones in the homozygous transgenic mice were specifically shorter and dimpled on their external surfaces. Chondrocytes in growth plates of all long bones were markedly disorganized and failed to align in columns. Analysis of growth plates from transgenic mice by in situ hybridization showed expression of alpha1(II) and alpha1(XI) but not of alpha1(I) or alpha1(V) which, in contrast, were expressed in the developing bone and in the bone collar. Expression of alpha1(X) specifically in the hypertrophic cartilage was observed in normal and transgenic mice. No obvious osteoarthritis was observed throughout the life of homozygous mice up to 1 year of age, although minor morphologic anomalies in the articular cartilages were discernible. The mild phenotype is consistent with similar mutations in the COL11A2 gene seen in patients with nonocular Stickler syndrome and some patients with otospondylomegaepiphyseal dysplasia (OSMED), as well as in patients with a nonsyndromic form of deafness called DFNA13.

Rat marrow stromal cells rapidly transduced with a self-inactivating retrovirus synthesize L-DOPA in vitro.

Autologous bone marrow stromal cells engineered to produce 3,4,-dihydroxyphenylalanine (L-DOPA) can potentially be used as donor cells for neural transplantation in Parkinson's disease. Here, we examined the possibility of using several different promoters and either a self-inactivating retrovirus (pSIR) or standard retroviruses to introduce into marrow stromal cells (MSCs), the two genes necessary for the cells to synthesize L-DOPA. pSIR vectors were constructed using the mouse phosphoglycerate kinase-1 (PGK) promoter or the cytomegalovirus (CMV) promoter to drive expression of either a GFP reporter gene or a bicistronic sequence containing the genes for human tyrosine hydroxylase type I (TH) and rat GTP cyclohydrolase I (GC) separated by an internal ribosome entry site (IRES). rMSCs were successfully transduced with both standard retroviral vectors and pSIR containing the PGK promoter. Transduced rMSCs expressed GFP (90.4--94.4% of cells) or were able to synthesize and secrete L-DOPA (89.0--283 pmols/10(6) cells/h). After transduced rMSCs were plated at low density (3--6 cells/cm(2)), the cells expanded over 1000-fold in 3--4 weeks, and the rMSCs continued to either express GFP or produce L-DOPA. Furthermore, two high-expressing clones were isolated and expanded at low-density from rMSCs transduced with pSIR driven by the PGK promoter (97.0% GFP+ or 1096.0 pmols L-DOPA/10(6) cells/h).

BMP-6 enhances chondrogenesis in a subpopulation of human marrow stromal cells.

Marrow stromal cells (MSCs) can differentiate into several mesenchymal lineages. MSCs were recently shown to form cartilage in micromass cultures with serum-free medium containing TGF-beta and dexamethasone. Here we found that addition of BMP-6 increased the weight of the pellets about 10-fold and they stained more extensively for proteoglycans. mRNAs for type II procollagen and type X collagen were detected at 1 week and the levels were increased at 3 weeks. We also compared two subpopulation of cultures of MSCs: Small and rapidly self-renewing cells (RS cells) and the large, more mature and slowly replicating cells (mMSCs). The cartilage pellets prepared from cultures enriched for RS cells were about 2.5-fold larger, stained more extensively for proteoglycans, and had levels of mRNA for type II procollagen that were 1.6-fold higher. Also, RS cells retained more of their chondrogenic potential as the cells were passaged.

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.

Rat marrow stromal cells are more sensitive to plating density and expand more rapidly from single-cell-derived colonies than human marrow stromal cells.

Human marrow stromal cell (hMSCs) were recently shown to expand rapidly in culture when plated at a low density of approximately 3 cells/cm(2). Low-density plating promoted proliferation of small recycling stem (RS) cells that appeared to be the most multipotent cells in the cultures. Here we demonstrated that MSCs from rat bone marrow (rMSCs) are even more sensitive to low-density plating than hMSCS: When plated at approximately 2 cells/cm(2), the cells expanded over 4,000-fold in 12 days, over twice the maximal rate observed with hMSCS: Analysis by fluorescence-activated cell sorter demonstrated that rMSCs had the same heterogeneity seen with hMSCs in that the cultures contained both small rapidly RS cells and much larger mature cells (mMSCs). The rat mMSCs differed from human mMSCs in that they regenerated RS cells in culture. Also, after low-density plating, colonies of rMSCs expanded into confluent cultures, whereas colonies of hMSCs did not.

Transgenic mice with inactive alleles for procollagen N-proteinase (ADAMTS-2) develop fragile skin and male sterility.

Transgenic mice were prepared with inactive alleles for procollagen N-proteinase (ADAMTS-2; where ADAMTS stands for a disintegrin and metalloproteinase with thrombospondin repeats). Homozygous mice were grossly normal at birth, but after 1-2 months they developed thin skin that tore after gentle handling. Although the gene was inactivated, a large fraction of the N-propeptides of type I procollagen in skin and the N-propeptides of type II procollagen in cartilage were cleaved. Therefore the results suggested the tissues contained one or more additional enzymes that slowly process the proteins. Electron microscopy did not reveal any defects in the morphology of collagen fibrils in newborn mice. However, in two-month-old mice, the collagen fibrils in skin were seen as bizarre curls in cross-section and the mean diameters of the fibrils were approx. half of the controls. Although a portion of the N-propeptides of type II procollagen in cartilage were not cleaved, no defects in the morphology of the fibrils were seen by electron microscopy or by polarized-light microscopy. Female homozygous mice were fertile, but male mice were sterile with a marked decrease in testicular sperm. Therefore the results indicated that ADAMTS-2 plays an essential role in the maturation of spermatogonia.

More knee joint osteoarthritis (OA) in mice after inactivation of one allele of type II procollagen gene but less OA after lifelong voluntary wheel running exercise.

OBJECTIVE: To investigate the incidence and severity of osteoarthritis (OA) and the effects of voluntary wheel running in normal mice and mice carrying either a targeted inactivation of one allele, heterozygous 'knockout', of Col2a1 gene or both alleles, homozygous 'knockout', of Col11a2 gene. METHODS: Mice lived until 15 months of age in individual cages. Running activity was recorded around the clock. OA changes were evaluated from serial knee joint sections by light microscopy. RESULTS: Heterozygous inactivation of Col2a1 gene coding for type II procollagen made the cartilage more susceptible to OA. At 15 months of age, OA prevalence was 60-90% in knockouts and 20-45% in normal controls (P < 0.01-0.001). Unexpectedly, a reduction of OA due to wheel running was observed in both knockout strains (P< 0.05-0.01). This effect was most evident in the femoral condyles. Incidence of OA in runners was approximately 50-85% of that in sedentary littermates. OA prevalence was higher in normal control and runner mice with high body weight. Running did not affect OA development in normal mice. CONCLUSION: Heterozygous knockout of Col2a1 gene increased the OA prevalence in mice. Lifelong voluntary wheel running had a protective effect against OA in both knockout mice lines. The reason for this remains unknown. Reduction of OA may result from the reorganization and strengthening of the articular cartilage collagen network and/or adjacent muscles due to running, or lower body weight. Increased compliance of the articular cartilage and bones of the knockout mice may also contribute to the reduction of OA in exercised animals.

In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP.

Human marrow stromal cells (hMSCs) are multipotential stem cells that can be differentiated into bone, cartilage, fat, and muscle. In the experiments here, we found that undifferentiated cultures of hMSCs express some markers characteristic of neural cells such as microtubule-associated protein 1B (MAP1B), neuron-specific tubulin (TuJ-1), neuron-specific enolase (NSE), and vimentin. By treating hMSCs with 0.5 mM isobutylmethylxanthine (IBMX)/1 mM dibutyryl cyclic AMP (dbcAMP) for 6 days, about 25% of the hMSCs differentiated into cells with a typical neural cell morphology and with increased levels of both NSE and vimentin. The data suggested that the hMSCs may have been differentiated into early progenitors of neural cells in vitro under conditions that increase the intracellular level of cAMP.

Inactivation of one allele of the type II collagen gene alters the collagen network in murine articular cartilage and makes cartilage softer.

OBJECTIVE: To evaluate the influence of inactivation of one allele ("heterozygous knockout" or "heterozygous inactivation") of the type II procollagen gene (Col2a1) on the biomechanical properties and structure of the articular cartilage and subchondral bone in 15 month old mice. METHODS: Indentation stiffness of the humerus head articular cartilage was measured by a microindentation method. Cartilage and subchondral bone were prepared for digital densitometry of proteoglycans (PGs), polarised light microscopy (PLM) of collagen, and osteoarthrosis (OA) grading. RESULTS: Heterozygous inactivation of the Col2a1 gene softened articular cartilage (p=0.002) as measured by indentation stiffness ((mean (SEM) 0.50 (0.07) MPa v 0.94 (0.13) MPa in controls). Fibrillar collagen network exhibited lower birefringence in the intermediate (p=0.04) and deep zones (p=0.01) of cartilage by PLM, indicating either decreased collagen content or a lower degree of fibril parallelism in the knockout mice. The total and zonal thicknesses of articular cartilage were unchanged. Zonal PG contents did not differ significantly. In knockout mice, the prevalence of superficial fibrillation-that is, a sign of OA, was higher than in controls (73% v 21%, p=0.002). The collagen induced birefringence of the superficial zone was not reduced. The subchondral bone volume fraction was lower in knockout mice than in controls, 31% v 43% (p=0.01), and optical retardation values in PLM of bone collagen were slightly but significantly lower (p=0.01). CONCLUSION: Heterozygous inactivation of the Col2a1 gene made articular cartilage softer, altered the collagenous network, reduced subchondral bone volume, and altered its microstructure. Changes in the cartilage collagen network probably contributed to increased susceptibility to OA.