Scientific Resources

Bone marrow-derived mesenchymal stem cells (MSC) have been defined as primitive,undifferentiated cells,capable of self-renewal and with the ability to give rise to different cell lineages,including adipocytes,osteocytes,fibroblasts,chondrocytes,and myoblasts. MSC are key components of the hematopoietic microenvironment. Several studies,including some from our own group,suggest that important quantitative and functional alterations are present in the stroma of patients with myelodysplasia (MDS). However,in most of such studies the stroma has been analyzed as a complex network of different cell types and molecules,thus it has been difficult to identify and characterize the cell(s) type(s) that is (are) altered in MDS. In the present study,we have focused on the biological characterization of MSC from MDS. As a first approach,we have quantified their numbers in bone marrow,and have worked on their phenotypic (morphology and immunophenotype) and cytogenetic properties. MSC were obtained by a negative selection procedure and cultured in a MSC liquid culture medium. In terms of morphology,as well as the expression of certain cell markers,no differences were observed between MSC from MDS patients and those derived from normal marrow. In both cases,MSC expressed CD29,CD90,CD105 and Prolyl-4-hydroxylase; in contrast,they did not express CD14,CD34,CD68,or alkaline phosphatase. Interestingly,in five out of nine MDS patients,MSC developed in culture showed cytogenetic abnormalities,usually involving the loss of chromosomal material. All those five cases also showed cytogenetic abnormalities in their hematopoietic cells. Interestingly,in some cases there was a complete lack of overlap between the karyotypes of hematopoietic cells and MSC. To the best of our knowledge,the present study is the first in which a pure population of MSC from MDS patients is analyzed in terms of their whole karyotype and demonstrates that in a significant proportion of patients,MSC are cytogenetically abnormal. Although the reason of this is still unclear,such alterations may have an impact on the physiology of these cells. Further studies are needed to assess the functional integrity of MDS-derived MSC. View Publication

Embryonic stem (ES) cells have the potential to serve as an alternative source of hematopoietic precursors for transplantation and for the study of hematopoietic cell development. Using coculture of human ES (hES) cells with OP9 bone marrow stromal cells,we were able to obtain up to 20% of CD34+ cells and isolate up to 10(7) CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8 to 9 days of culture. The hES cell-derived CD34+ cells were highly enriched in colony-forming cells,cells expressing hematopoiesis-associated genes GATA-1,GATA-2,SCL/TAL1,and Flk-1,and retained clonogenic potential after in vitro expansion. CD34+ cells displayed the phenotype of primitive hematopoietic progenitors as defined by co-expression of CD90,CD117,and CD164,along with a lack of CD38 expression and contained aldehyde dehydrogenase-positive cells as well as cells with verapamil-sensitive ability to efflux rhodamine 123. When cultured on MS-5 stromal cells in the presence of stem cell factor,Flt3-L,interleukin 7 (IL-7),and IL-3,isolated CD34+ cells differentiated into lymphoid (B and natural killer cells) as well as myeloid (macrophages and granulocytes) lineages. These data indicate that CD34+ cells generated through hES/OP9 coculture display several features of definitive hematopoietic stem cells. View Publication

Differentiating embryonic stem (ES) cells are an increasingly important source of hematopoietic progenitors,useful for both basic research and clinical applications. Besides their characterization in colony assays,protocols exist for the cultivation of lymphoid,myeloid,and erythroid cells. With the possible exception of mast cells,however,long-term expansion of pure hematopoietic progenitors from ES cells has not been possible without immortalization caused by overexpression of exogenous genes. Here,we describe for the first time an efficient yet easy strategy to generate mass cultures of pure,immature erythroid progenitors from mouse ES cells (ES-EPs),using serum-free medium plus recombinant cytokines and hormones. ES-EPs represent long-lived,adult,definitive erythroid progenitors that resemble immature erythroid cells expanding in vivo during stress erythropoiesis. When exposed to terminal differentiation conditions,ES-EPs differentiated into mature,enucleated erythrocytes. Importantly,ES-EPs injected into mice did not exhibit tumorigenic potential but differentiated into normal erythrocytes. Both the virtually unlimited supply of cells and the defined culture conditions render our system a valuable tool for the analysis of factors influencing proliferation and maturation of erythroid progenitors. In addition,the system allows detailed characterization of processes during erythroid proliferation and differentiation using wild-type (wt) and genetically modified ES cells. View Publication

We have observed karyotypic changes involving the gain of chromosome 17q in three independent human embryonic stem (hES) cell lines on five independent occasions. A gain of chromosome 12 was seen occasionally. This implies that increased dosage of chromosome 17q and 12 gene(s) provides a selective advantage for the propagation of undifferentiated hES cells. These observations are instructive for the future application of hES cells in transplantation therapies in which the use of aneuploid cells could be detrimental. View Publication

The physiologic function of BUBR1,a key component of the spindle checkpoint,was examined by generating BUBR1-mutant mice. BUBR1(-/-) embryos failed to survive beyond day 8.5 in utero as a result of extensive apoptosis. Whereas BUBR1(+/-) blastocysts grew relatively normally in vitro,BUBR1(-/-) blastocysts exhibited impaired proliferation and atrophied. Adult BUBR1(+/-) mice manifested splenomegaly and abnormal megakaryopoiesis. BUBR1 haploinsufficiency resulted in an increase in the number of splenic megakaryocytes,which was correlated with an increase in megakaryocytic,but a decrease in erythroid,progenitors in bone marrow cells. RNA interference-mediated down-regulation of BUBR1 also caused an increase in polyploidy formation in murine embryonic fibroblast cells and enhanced megakaryopoiesis in bone marrow progenitor cells. However,enhanced megakaryopoiesis in BUBR1(+/-) mice was not correlated with a significant increase in platelets in peripheral blood,which was at least partly due to a defect in the formation of proplatelet-producing megakaryocytes. Together,these results indicate that BUBR1 is essential for early embryonic development and normal hematopoiesis. View Publication

Embryonic stem cells can be induced in vitro,by coculture with the stromal line RP.0.10 and a mixture of interleukins 3,6,and 7,to differentiate into T (Joro75+) and B (B-220+) lymphocyte progenitors and other (Thy-1+,PgP-1+,c-kit+,Joro75-,B-220-,F4/80-,Mac-1-) hemopoietic precursors. The progeny of in vitro-induced embryonic stem cells can reconstitute the lymphoid compartments of T- and B-lymphocyte-deficient scid mice and generate mature T and B lymphocytes in sublethally irradiated normal mice. Exogenous cytokines can dramatically alter the developmental fate of embryonic stem cells in culture. The in vitro system described here should facilitate the study of molecular events leading to cell-lineage commitment and to the formation of hemopoietic stem cells and their immediate lymphoid progeny. View Publication

Undifferentiated pluripotent stem cells with flexible developmental potentials are not normally found in peripheral blood. However,such cells have recently been reported to reside in the bone marrow. Herein are reported methods of inducing pluripotency in cells derived from unmobilised adult human peripheral blood. In response to the inclusion of purified CR3/43 monoclonal antibody (mAb) to well-established culture conditions,mononuclear cells (MNC) obtained from a single blood donor are converted into pluripotent haematopoietic,neuronal and cardiomyogenic progenitor stem cells or undifferentiated stem cells. The haematopoietic stem cells are CD34+,clonogenic and have been shown to repopulate non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The neuronal precursors transcribe the primitive stem cell markers OCT-4 and nestin,and on maturation,differentially stain positive for neuronal,glial or oligodendrocyte-specific antigens. The cardiomyogenic progenitor stem cells form large bodies of asynchronously beating cells and differentiate into mature cardiomyocytes which transcribe GATA-4. The undifferentiated stem cells do not express haematopoietic-associated markers,are negative for major histocompatibility complex (MHC) class I and II antigens,transcribe high levels of OCT-4 and form embryoid body (EB)-like structures. This induction of stem cell-like plasticity in MNC may have proceeded by a process of retrodifferentiation but,in any case,could have profound clinical and pharmacological implications. Finally,the flexibility and the speed by which a variety of stem cell classes can be generated ex vivo from donor blood could potentially transfer this novel process into a less invasive automated clinical procedure. View Publication

Differentiation of pluripotent embryonic stem (ES) cells is associated with expression of fate-specifying gene products. Coordinated development,however,must involve modifying factors that enable differentiation and growth to adjust in response to local microenvironmental determinants. We report here that the ephrin receptor,EphB4,known to be spatially restricted in expression and critical for organized vessel formation,modifies the rate and magnitude of ES cells acquiring genotypic and phenotypic characteristics of mesodermal tissues. Hemangioblast,blood cell,cardiomyocyte,and vascular differentiation was impaired in EphB4-/- ES cells in conjunction with decreased expression of mesoderm-associated,but not neuroectoderm-associated,genes. Therefore,EphB4 modulates the response to mesoderm induction signals. These data add differentiation kinetics to the known effects of ephrin receptors on mammalian cell migration and adhesion. We propose that modifying sensitivity to differentiation cues is a further means for ephrin receptors to contribute to tissue patterning and organization. View Publication

The signal transducer Stat5 plays a key role in the regulation of hematopoietic differentiation and hematopoietic stem cell function. To evaluate the effects of Stat5 signaling in the earliest hematopoietic progenitors,we have generated an embryonic stem cell line in which Stat5 signaling can be induced with doxycycline. Ectopic Stat5 activation at the point of origin of the hematopoietic lineage (from day 4 to day 6 of embryoid body differentiation) significantly enhances the number of hematopoietic progenitors with colony-forming potential. It does so without significantly altering total numbers or apoptosis of hematopoietic cells,suggesting a cell-intrinsic effect of Stat5 on either the developmental potential or clonogenicity of this population. From day-6 embryoid bodies,under the influence of Stat5 signaling,a population of semiadherent cells can be expanded on OP9 stromal cells that is comprised of primitive hematopoietic blast cells with ongoing,mainly myeloid,differentiation. When these cells are injected into lethally irradiated mice,they engraft transiently in a doxycycline-dependent manner. These results demonstrate that the hematopoietic commitment of embryonic stem cells may be augmented by a Stat5-mediated signal,and highlight the utility of manipulating individual components of signaling pathways for engineering tissue-specific differentiation of stem cells. View Publication

Human stem cells derived from human fertilized oocytes,fetal primordial germ cells,umbilical cord blood,and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However,the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach,employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D),we established stem cell lines homozygous for H-2-B and H-2-D,respectively. The undifferentiated cells retained a normal karyotype,expressed stage-specific embryonic antigen-1 and Oct4,and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition,these cells demonstrated the potential for in vitro differentiation into endoderm,neuronal,and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes,and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation� View Publication

Hematopoietic stem cells (HSC) are tightly regulated through,as yet,undefined mechanisms that balance self-renewal and differentiation. We have identified a role for the transcriptional coactivators CREB-binding protein (CBP) and p300 in such HSC fate decisions. A full dose of CBP,but not p300,is crucial for HSC self-renewal. Conversely,p300,but not CBP,is essential for proper hematopoietic differentiation. Furthermore,in chimeric mice,hematologic malignancies emerged from both CBP(-/-) and p300(-/-) cell populations. Thus,CBP and p300 play essential but distinct roles in maintaining normal hematopoiesis,and,in mice,both are required for preventing hematologic tumorigenesis. View Publication

Embryonic stem (ES) cells have been established as permanent lines of undifferentiated pluripotent cells from early mouse embryos. ES cells provide a unique system for the genetic manipulation and the creation of knockout strains of mice through gene targeting. By cultivation in vitro as 3D aggregates called embryoid bodies,ES cells can differentiate into derivatives of all 3 primary germ layers,including cardiomyocytes. Protocols for the in vitro differentiation of ES cells into cardiomyocytes representing all specialized cell types of the heart,such as atrial-like,ventricular-like,sinus nodal-like,and Purkinje-like cells,have been established. During differentiation,cardiac-specific genes as well as proteins,receptors,and ion channels are expressed in a developmental continuum,which closely recapitulates the developmental pattern of early cardiogenesis. Exploitation of ES cell-derived cardiomyocytes has facilitated the analysis of early cardiac development and has permitted in vitro gain-of-function" or "loss-of-function" genetic studies. Recently� View Publication