Effective repair of articular cartilage using human pluripotent stem cell-derived tissue.

In an effort to develop an effective source of clinically relevant cells and tissues for cartilage repair a directed differentiation method was used to generate articular chondrocytes and cartilage tissues from human embryonic stem cells (hESCs). It has previously been demonstrated that chondrocytes derived from hESCs retain a stable cartilage-forming phenotype following subcutaneous implantation in mice. In this report, the potential of hESC-derived articular-like cartilage to repair osteochondral defects created in the rat trochlea was evaluated. Articular cartilage-like tissues were generated from hESCs and implanted into the defects. After 6 and 12 weeks, the defects were evaluated histologically and immunohistochemically, and the quality of repair was assessed using a modified ICRS II scoring system. Following 6 and 12 weeks after implantation, hESC-derived cartilage tissues maintained their proteoglycan and type II collagen-rich matrix and scored significantly higher than control defects, which had been filled with fibrin glue alone. Implants were found to be well integrated with native host tissue at the basal and lateral surfaces, although implanted human cells and host cells remained regionally separated. A subset of implants underwent a process of remodeling similar to endochondral ossification, suggesting the potential for a single cartilaginous implant to promote the generation of new subchondral bone in addition to repair of the articular cartilage. The ability to create cartilage tissues with integrative and reparative properties from an unlimited and robust cell source represents a significant advance for cartilage repair that can be further developed in large animal models before clinical- setting application.

Directed differentiation of pluripotent stem cells: from developmental biology to therapeutic applications.

The discovery of human pluripotent stem cells has laid the foundation for an emerging new field of biomedical research that holds promise to develop models of human development and disease, establish new strategies for discovering and testing drugs, and provide systems for the generation of cells and tissues for transplantation for the treatment of disease. The remarkable potential of pluripotent stem cells has sparked interest and excitement in academia, the biotechnology and pharmaceutical industries, as well as the lay public. Although the potential of human pluripotent stem cells is truly outstanding, fulfilling this potential is solely dependent on our ability to efficiently generate functional cell types from them. Some of the most successful approaches in this area to date are those that have applied the principles of developmental biology to stem cell differentiation. In this chapter, we review these concepts and highlight specific examples demonstrating that pluripotent stem cell differentiation in culture recapitulates the key aspects of early embryonic development. By continuing to translate insights from embryology to stem cell biology, progress in our ability to generate specific cell types from pluripotent stem cells will advance, yielding enriched populations of human cell types, including cardiomyocytes, hematopoietic cells, hepatocytes, pancreatic beta cells, and neural cells, for drug discovery, functional evaluation in preclinical models of human disease, and ultimately clinical applications.

Genomic instability-based transgenic models of prostate cancer.

To develop animal models that represent the broad spectrum of human prostate cancer, we created transgenic mice with targeted prostate-specific expression of two genes (ECO:RI and c-fos) implicated in the induction of genomic instability. Expression of the transgenes was restricted to prostate epithelial cells by coupling them to the tissue-specific, hormonally regulated probasin promoter (PB). The effects of transgene expression were examined histologically in prostate sections at time points taken from 4 to 24 months of age. The progressive presence of regions of mild-to-severe hyperplasia, low- and high-grade prostatic intra-epithelial neoplasia, and well-differentiated adenocarcinoma was observed in both PBECO:RI lines but no significant pathology was detected in the PBfos line. Prostate tissue of PBECO:RI mice was examined for expression of p53, proliferating cell nuclear antigen (PCNA) and Ki67 at multiple time points. Although p53 does not appear to be mutated, levels of PCNA and Ki67 are elevated and correlate with the severity of the prostatic lesions. Overall, pre-neoplastic and neoplastic stages represented in the PBECO:RI model showed similarity to corresponding early stages of the human disease. This genomic instability-based model will be used to study the mechanisms involved in the early stages of prostate carcinogenesis and to investigate the nature of subsequent events necessary for the progression to advanced disease.

Mitogen-activated protein kinase activation through Fc epsilon receptor I and stem cell factor receptor is differentially regulated by phosphatidylinositol 3-kinase and calcineurin in mouse bone marrow-derived mast cells.

Aggregation of high affinity FcR for IgE (Fc epsilon RI) on mast cells activates intracellular signal transduction pathways, including the activation of protein tyrosine kinases, phosphatidylinositol 3-kinase (PI3-kinase), and protein kinase C. Binding of stem cell factor (SCF) to its receptor (SCFR, c-Kit) on mast cells also induces increases in intrinsic tyrosine kinase activity and activation of PI3-kinase. Although ligation of both receptors induces Ras and Raf-1 activation, the downstream consequences of these early activation events are not well defined, except for the activation of extracellular signal-regulated kinases (ERK). Addition of Ag (OVA) to mouse bone marrow-derived mast cells (BMMC) sensitized with anti-OVA IgE triggers the activation of three members of the mitogen-activated protein (MAP) kinase family, c-Jun amino-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinases. SCF similarly activates all three MAP kinases. Wortmannin, an inhibitor of PI3-kinase, inhibited both Fc epsilon RI- and SCFR-mediated JNK activation and partially inhibited Fc epsilon RI, but not SCFR-mediated p38 activation. Cyclosporin A inhibited Fc epsilon RI-mediated JNK and p38 activation, but did not affect the activation of these kinases when stimulated through the SCFR. Wortmannin and cyclosporin A inhibited Fc epsilon RI-mediated production of TNF-alpha and IL-4 in addition to serotonin release in BMMC. These results indicate that both PI3-kinase and calcineurin may contribute to the regulation of cytokine gene transcription and the degranulation response by modulating JNK activity in BMMC.

Stem cell factor augments Fc epsilon RI-mediated TNF-alpha production and stimulates MAP kinases via a different pathway in MC/9 mast cells.

Mast cells express the receptor tyrosine kinase kit/stem cell factor receptor (SCFR) which is encoded by the proto-oncogene c-kit. Ligation of SCFR induces its dimerization and activation of its intrinsic tyrosine kinase activity leading to activation of Raf-1, phospholipases, phosphatidylinositol 3-kinase, and extracellular signal-regulated kinases. However, little is known about the downstream signals initiated by SCFR ligation except for activation of extracellular signal-regulated kinases. The murine mast cell line, MC/9, synthesizes and secretes TNF-alpha following the aggregation of high affinity Fc receptors for IgE (Fc epsilonRI). Ligation of SCFR or Fc epsilonRI on MC/9 cells resulted in the activation of all three MAP kinase family members, extracellular signal-regulated kinases, c-Jun amino-terminal kinase (JNK), and p38. Stem cell factor (SCF)-induced activation of JNK and p38 was insensitive to wortmannin, cyclosporin A, and FK506 whereas activation of these kinases through Fc epsilonRI was sensitive to these drugs. Coligation of SCFR augmented Fc epsilonRI-mediated activation of MAP kinases, especially JNK activation, and SCF augmented Fc epsilonRI-mediated TNF-alpha production in MC/9 cells, although SCF alone did not induce TNF-alpha production. This augmentation by SCF was regulated at the level of transcription, at least in part, since the promoter activity of TNF-alpha was enhanced following addition of SCF. These results demonstrate that SCF can augment Fc epsilonRI-mediated JNK activation and cytokine gene transcription but via pathways that are regulated differently than the ones activated through Fc epsilonRI.

Targeted disruption of p70(s6k) defines its role in protein synthesis and rapamycin sensitivity.

Here, we disrupted the p70 S6 kinase (p70(s6k)) gene in murine embryonic stem cells to determine the role of this kinase in cell growth, protein synthesis, and rapamycin sensitivity. p70(s6k-/-) cells proliferated at a slower rate than parental cells, suggesting that p70(s6k) has a positive influence on cell proliferation but is not essential. In addition, rapamycin inhibited proliferation of p70(s6k-/-) cells, indicating that other events inhibited by the drug, independent of p70(s6k), also are important for both cell proliferation and the action of rapamycin. In p70(s6k-/-) cells, which exhibited no ribosomal S6 phosphorylation, translation of mRNA encoding ribosomal proteins was not increased by serum nor specifically inhibited by rapamycin. In contrast, rapamycin inhibited phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1), general mRNA translation, and overall protein synthesis in p70(s6k-/-) cells, indicating that these events proceed independently of p70(s6k) activity. This study localizes the function of p70(s6k) to ribosomal biogenesis by regulating ribosomal protein synthesis at the level of mRNA translation.

Macrocerebellum: neuroimaging and clinical features of a newly recognized condition.

Other than hamartomatous enlargement of the cerebellum as in Lhermitte-Duclos syndrome, diffuse enlargement of the cerebellum is not clearly described. We report four patients (ages 9 months to 2 years) with diffusely enlarged cerebelli as identified by measurement of the cerebellum and comparison to age appropriate normal values. The cerebellar measurements were determined in absolute numbers and expressed as ratios of cerebellum to whole brain and supratentorial brain. The clinical features of these four children (3 boys, 1 girl) consistently include global developmental delay, tone abnormalities, preserved reflexes, delayed or abnormal maturation of the visual system (oculomotor apraxia), and deficient or delayed myelination of cerebral white matter. The etiology of the macrocerebellum is unknown but we propose that the cerebellum is responding to the elaboration of growth factors intended to augment the slow development of cerebral structures. Regardless of the etiology, the finding of a macrocerebellum appears to allow the clinician to predict the clinical features of the patient and probably represents a marker for disturbed cerebral development.

Size of the corpus callosum in cerebral palsy.

It has been suggested that the size of the corpus callosum may have diagnostic significance in cerebral palsy, although this relationship is incompletely defined. Ninety-one patients with cerebral palsy had been studied by magnetic resonance imaging in the 5-year period from 1990 to 1994. Fifty-seven of these 91 patients had a technically appropriate midsagittal magnetic resonance image for quantitative morphometric analysis. The ratio of the area of the corpus callosum to the area of the supratentorial brain was compared to published age- and gender-specific norms. Imaging findings were correlated with clinical history and cause of cerebral palsy. The corpus callosum was of normal size in 43 patients and more than 2 standard deviations below the mean in 14 patients. The causes for cerebral palsy included hypoxic ischemic encephalopathy (32), cerebral dysgenesis (8), and porencephalic strokes (6); the etiology could not be established in 11 patients. The size of the corpus callosum was highly correlated with the cause of cerebral palsy, such that all patients with cerebral dysgenesis had hypoplasia of the corpus callosum (one-sided z test, p < 0.0001). Conversely, the callosum was of normal size in 32 of 38 patients with hypoxic ischemic encephalopathy and porencephalic strokes. The presence of a hypoplastic corpus callosum is highly associated with cerebral dysgenesis as a cause for cerebral palsy.

In vitro differentiation of embryonic stem cells.

Under appropriate conditions in culture, embryonic stem cells will differentiate and form embryoid bodies that have been shown to contain cells of the hematopoietic, endothelial, muscle and neuronal lineages. Many aspects of the lineage-specific differentiation programs observed within the embryoid bodies reflect those found in the embryo, indicating that this model system provides access to early cell populations that develop in a normal fashion. Recent studies involving the differentiation of genetically altered embryonic stem cells highlight the potential of this in vitro differentiation system for defining the function of genes in early development.

Product inhibition of benzo[a]pyrene metabolism in uninduced rat liver microsomes: effect of diol epoxide formation.

Conversion of benzo[a]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770-13776). Competition between BP and 7,8-dihydrodiol for P-450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4-6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2-25 pmol/mg) of the very active cytochrome P-450c, which is the predominant form in MC-microsomes. The major effect of elevated levels of P-450c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of Km (7.9-2.6 microM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5-15% of total BP metabolites). The formation of DE was directly correlated with the content of P-450c (r = 0.94). The presence of increased levels of P-450c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.

Generation of purified stromal cell cultures that support lymphoid and myeloid precursors.

Treatment of Dexter-type long-term bone marrow cultures with the antibiotic mycophenolic acid (MPA) eliminates all hemopoietic cells from the cultures, while a morphologically intact, adherent stromal cell layer is retained. The ability of these MPA treated stromal cell cultures to support long-term hemopoiesis was tested by seeding them with fresh bone marrow cells that had been passed through nylon wool. This procedure yields a relatively stromal cell depleted population of hemopoietic cells. An aliquot of 5 X 10(5) or 2.5 X 10(5) nylon wool passed bone marrow cells bearing the T6 chromosomal marker was seeded onto replicate MPA-treated stromal cell layers. The stromal cells stimulated the proliferation of the bone marrow cells, and nonadherent cells were present for up to 8 weeks of culture. Progenitors of granulocytes and macrophages (CFU-GM) were also present for this period of time despite weekly demi-depopulation, during culture feeding. Karyotypic analysis confirmed that the CFU-GM were derived from the reseeded population. Nylon wool-passed bone marrow cells seeded alone into empty flasks under identical conditions did not survive past 1 week. Cells from the reseeded cultures were also tested for early myeloid precursors (CFU-S) and injected into immunodeficient CBA/N mice to test for the presence of primitive B cell precursors. CFU-S were present in mice killed 11 days following injection of cells, and high levels of B cell colony-forming units (CFU-B) were present in mice 4 weeks post reconstitution. Further studies demonstrated that factors present in medium conditioned by the stromal cells could support the growth of CFU-GM. These data indicate that treatment of long-term bone marrow cultures with MPA results in a population of functional stromal cells.

Maintenance of hemopoiesis in long-term bone marrow cultures from S1/S1d and W/Wv mice.

Although phenotypically similar, the cellular defects in congenitally anemic mice of genotype W/Wv and S1/S1d are quite different. W/Wv mice have defective hemopoietic stem cells; in contrast, S1/S1d mice have normal stem cells, but their hemopoietic microenvironment cannot support normal differentiation of the stem cells. We also observed defective hemopoiesis as measured by granulocyte-macrophage colony-forming units (GM-CFU) in long-term bone marrow cultures (LTBMC) established with marrow from these mutants, but in contrast to an earlier report we obtained long-term maintenance of hemopoiesis (up to 20 weeks) albeit at a lower level than the control (28% of control for S1/S1d and 23% for W/Wv). These levels probably reflect more accurately the in vivo effects of the mutations than the severe defect reported previously. However, this level of hemopoiesis and the high variability observed in replicate flasks in LTBMC make it difficult to study these mutants in tissue culture.

Benzo(a)pyrene activation to 7,8-dihydrodiol 9,10-oxide by rat liver microsomes. Control by selective product inhibition.

Metabolism of benzo(a)pyrene (BP) and 7,8-dihydrodiol by 3-methylcholanthrene (MC)-induced rat liver microsomes are both subject to severe inhibition by primary metabolites of BP, which was analyzed by determining individual inhibition constants for all primary BP metabolites for both BP and 7,8-dihydrodiol metabolism. Monooxygenation of 7,8-dihydrodiol was, surprisingly, 5 to 10 times more sensitive than monooxygenation of BP to inhibition by all primary metabolites, even though both reactions require the same enzyme, cytochrome P-450c. Two representative products, 1,6-quinone and 9-phenol, were both strong, competitive inhibitors of BP metabolism with Ki values of 0.12 and 0.74 microM, respectively. The total effect of product inhibition on the overall reactions was determined by fitting progress curves of BP, 7,8-dihydrodiol, and anti-7,8-dihydrodiol 9,10-oxide (determined as 7,10/8,9-tetrol) over a range of BP concentrations to integrated steady-state equations using experimental Vmax and Km values. The effective product inhibition factors for BP and 7,8-dihydrodiol metabolism, determined from progress curve fits, were only 2-fold higher than the corresponding calculated theoretical values. The effective product inhibition factors, obtained from progress curve analysis, confirmed that 7,8-dihydrodiol metabolism was substantially more sensitive to inhibition by primary BP metabolites than BP metabolism itself. This difference probably reflects the much higher affinity of cytochrome P-450c for BP (Kd = 6 nM), as compared to 7,8-dihydrodiol (Kd = 175 nM) that was established spectrophotometrically both for the purified cytochrome and for MC microsomes. The Km for BP metabolism is 50 to 100 times higher than the Kd, while the Km is similar to the Kd for 7,8-dihydrodiol metabolism. The discrepancy for BP between Km and Kd suggests that standard Michaelis-Menten kinetics may be perturbed by either slow substrate or product dissociation.

Functional status of cells from lymphoid and myeloid tissues in mice with severe combined immunodeficiency disease.

Cells from mice with severe combined immunodeficiency disease (SCID) were tested in assays that measure myeloid and lymphoid function. Results showed that C.B-17 scid and their normal counterparts (C.B-17) have similar levels of spleen colony-forming units. The frequency of in vitro myeloid colony-forming units in C.B-17 scid spleen is elevated, but the absolute number of colony-forming units in C.B-17 scid and C.B-17 spleen is similar. The absolute number of bone marrow colony-forming units in C.B-17 scid and C.B-17 mice is comparable. Cells from C.B-17 scid spleen are consistently negative in all tests of B and T cell function. C.B-17 scid splenocytes fail to proliferate in response to T and B cell mitogens or to allogeneic lymphocytes in a one-way MLR; C.B-17 scid cells do serve as stimulators in MLR. B lymphocyte colony-forming units are absent, as are cytotoxic lymphocyte precursors and cells that can generate T cell colonies with cytotoxic progenitors. The microenvironment of the C.B-17 scid mouse is conducive to lymphocyte differentiation, because functional B and T cells are easily detectable in mice reconstituted with normal bone marrow cells. The results of this study indicate that scid specifically impairs the differentiation of stem cells into mature lymphocytes; myeloid cell differentiation is not affected.

Hemopoiesis in spleen and bone marrow cultures.

Long-term cultures established from spleen cells were compared to those established from bone marrow cells for their ability to maintain hemopoiesis as measured by the presence of hemopoietic progenitor cells (in vitro CFU) and multipotent stem cells (CFU-S). The frequency of both in vitro CFU and CFU-S increased dramatically during the first 2 weeks in the spleen cultures. Following this early peak of activity, the number of progenitors and stem cells declined to undetectable levels by week 6 of culture. During this short phase of hemopoiesis, large amounts of GM-CSF could be detected in the supernatant of the spleen cultures. In contrast, bone marrow cultures did not share this early peak of hemopoiesis; however, they maintained activity for much longer periods of time than did the spleen cultures. When spleen stem cells were seeded onto functional bone marrow adherent cells, spleen-derived in vitro CFU were maintained well beyond week 6 of culture. Spleen cultures established from athymic nu/nu mice showed a greatly reduced ability to support hemopoiesis while those from S1/S1d mice maintained GM-CFU as well as cultures from normal mice.

Modulation of microsomal benzo[a]pyrene metabolism by DNA.

Inclusion of calf thymus DNA during microsomal benzo[a]pyrene (BP) metabolism increases product formation by decreasing the accessibility of microsomal enzymes to inhibitory BP quinones. The relief of product inhibition of BP metabolism, the stimulation of the formation of BP 7,8-dihydrodiol-9,10-oxides (DE), and the corresponding DNA adducts were all dependent to varying extents on DNA concentration. The role of BP quinones was evidenced by effects of DNA on all aspects of quinone reactivity: (a) inhibition of microsomal reduction of quinones, (b) inhibition of quinone glucuronidation, (c) inhibition of quinone monooxygenation, (d) a substantial reduction of the inhibition of BP metabolism and diol epoxide (DE) formation by added BP 6,12-quinone, and (e) a stimulation of BP metabolism even though quinine levels were also increased. DNA inhibited the reduction of 1,6- and 3,6-quinone to a similar degree under both oxygen-depleted and aerobic conditions. Other effects of DNA were very selective; glucuronidation of added 1,6- and 6,12-quinone was inhibited less than glucuronidation of 3,6-quinone (35% and 50% versus over 80%). However, monooxygenation of 3,6-quinone was not inhibited, whereas monooxygenation of 1,6-quinone was reduced by 60-70%. There was no measurable monooxygenation of 6,12-quinone. This specificity may indicate that DNA exerts its effect not simply by sequestering BP quinones. The interaction with DNA produced a distinct 25-nm red shift in the visible spectra of 1,6- and 3,6-quinone, while the change in the 6,12-quinone spectrum was less pronounced. RNA induced a similar red shift in the 1,6-quinone spectrum. Spectral measurements indicated binding of one molecule of 1,6-quinone per 50 DNA base pairs, while binding to RNA was 10-fold less extensive. The binding of 1,6-quinone to DNA was decreased by Mg2+, suggesting that 1,6-quinone binds by intercalation. DNA perturbs BP metabolism in a second way by increasing the ratio of 9-phenol to 9,10-dihydrodiol 4-fold. This is probably due to a DNA-catalyzed rearrangement of 9,10-oxide to 9-phenol. This effect contributes substantially to the greater sensitivity of the formation of 9-phenol 4,5-oxide-DNA adducts to the DNA concentration. It is evident from these data that, in addition to binding carcinogens covalently, DNA can affect the kinetics and product distribution of carcinogen metabolism. The high capacity of DNA to sequester BP quinones from cellular membranes is likely to be associated with additional DNA damage which may contribute to carcinogenesis.