Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture.

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated.

Small molecule-directed specification of sclerotome-like chondroprogenitors and induction of a somitic chondrogenesis program from embryonic stem cells.

Pluripotent embryonic stem cells (ESCs) generate rostral paraxial mesoderm-like progeny in 5-6 days of differentiation induced by Wnt3a and Noggin (Nog). We report that canonical Wnt signaling introduced either by forced expression of activated β-catenin, or the small-molecule inhibitor of Gsk3, CHIR99021, satisfied the need for Wnt3a signaling, and that the small-molecule inhibitor of BMP type I receptors, LDN193189, was able to replace Nog. Mesodermal progeny generated using such small molecules were chondrogenic in vitro, and expressed trunk paraxial mesoderm markers such as Tcf15 and Meox1, and somite markers such as Uncx, but failed to express sclerotome markers such as Pax1.

Regulation of pluripotent cell differentiation by a small molecule, staurosporine.

Research in the embryo and in culture has resulted in a sophisticated understanding of many regulators of pluripotent cell differentiation. As a consequence, protocols for the differentiation of pluripotent cells generally rely on a combination of exogenous growth factors and endogenous signalling. Little consideration has been given to manipulating other pathways to achieve pluripotent cell differentiation.

Comprehensive transcriptome and immunophenotype analysis of renal and cardiac MSC-like populations supports strong congruence with bone marrow MSC despite maintenance of distinct identities.

Cells resembling bone marrow mesenchymal stem cells (MSC) have been isolated from many organs but their functional relationships have not been thoroughly examined. Here we compared the immunophenotype, gene expression, multipotency and immunosuppressive potential of MSC-like colony-forming cells from adult murine bone marrow (bmMSC), kidney (kCFU-F) and heart (cCFU-F), cultured under uniform conditions. All populations showed classic MSC morphology and in vitro mesodermal multipotency.

Methods to analyze the homing efficiency and spatial distribution of hematopoietic stem and progenitor cells and their relationship to the bone marrow endosteum and vascular endothelium.

The tracking of immunofluorescent labeled hematopoietic stem and progenitor cells (HSC/HPC) within the bone marrow (BM) cavity allows the assessment of the regulatory processes involved in transendothelial migration, trans-marrow migration, and finally lodgement into the HSC niche. This is of interest as the extracellular and cellular components involved in the regulation of HSC quiescence and differentiation are still not completely understood. Homing of transplanted HSC is the first critical step in the interaction between HSC and the microenvironment of the BM.

Phenotypically identical hemopoietic stem cells isolated from different regions of bone marrow have different biologic potential.

Hemopoietic stem cells (HSCs) reside within a specified area of the bone marrow (BM) cavity called a "niche" that modulates HSC quiescence, proliferation, differentiation, and migration. Our previous studies have identified the endosteal BM region as the site for the HSC niche and demonstrated that hemopoietic stem and progenitor populations (HSPCs, LSK) isolated from different BM regions exhibit significantly different hemopoietic potential. In this study, we have analyzed subpopulations of LSK cells isolated from different regions of the BM and showed that CD150(+)CD48(-)LSK HSCs within the endosteal BM region have superior proliferative capacity and homing efficiency compared with CD150(+)CD48(-)LSK HSCs isolated from the central BM.

Endogenous lung stem cells: what is their potential for use in regenerative medicine?

Advances in stem cell technologies in recent years have generated considerable interest in harnessing the potential of adult and embryonic stem cells in regenerative medicine. Stem cell-based therapies are a particularly attractive option for the treatment of intractable lung diseases for which current therapies are essentially palliative. Proof-of-principle experiments in animal models demonstrate the efficacy of exogenous stem cells in mediating lung repair by attenuating fibrotic responses to injury, but also suggest that their ability to contribute to lung epithelial regeneration and repair is limited.

Collaboration between WNT and BMP signaling promotes hemoangiogenic cell development from human fibroblast-derived iPS cells.

Induced pluripotent stem (iPS) cells are generated by nuclear reprogramming of mature cells to a pluripotent state, and show biological properties of embryonic stem (ES) cells. The observation that human (h)ES cells generate hemoangiogenic progeny, defined by their high-level expression of KDR and low-level expression of PDGFRalpha (KDR(+)PDGFRalpha(lo)) via WNT and BMP signaling during 5-8 days of differentiation in a serum-free environment led us to address how hiPS cells give rise to hemoangiogenic progeny. In the presence of WNT3a, four hiPS cell lines derived from human skin fibroblasts commonly generated KDR(+) and/or PDGFRalpha(+) progeny by day 8 of differentiation.

Isolation of clonogenic, long-term self renewing embryonic renal stem cells.

A tissue stem cell should exhibit long-term self-renewal, clonogenicity and a capacity to differentiate into the tissue of origin. Such a postnatal renal stem cell has not been formally identified. The metanephric mesenchyme (MM) of the developing kidney gives rise to both the renal interstitium and the nephrons and is regarded as the progenitor population of the developing kidney.

Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung.

The role of lung epithelial stem cells in maintenance and repair of the adult lung is ill-defined, and their identity remains contentious because of the lack of definitive markers for their prospective isolation and the absence of clonogenic assays able to measure their stem/progenitor cell potential. In this study, we show that replication of epithelial-mesenchymal interactions in a previously undescribed matrigel-based clonogenic assay enables the identification of lung epithelial stem/progenitor cells by their colony-forming potential in vitro. We describe a population of EpCAM(hi) CD49f(pos) CD104(pos) CD24(low) epithelial cfus that generate colonies comprising airway, alveolar, or mixed lung epithelial cell lineages when cocultured with EpCAM(neg) Sca-1(pos) lung mesenchymal cells.

SCUBE1, a novel developmental gene involved in renal regeneration and repair.

Background: We have identified that a novel developmental gene and protein, SCUBE1, is expressed in endothelial cells and may play an important role in kidney regeneration.

Methods: The temporal and spatial expression of SCUBE1 was determined in a mouse model of ischaemia-reperfusion (IR) injury at 3 days and 1, 3 and 6 weeks post-injury by immunofluorescence microscopy. In vitro analysis was used to examine SCUBE1 signalling in endothelial cells under conditions of cell stress using quantitative real-time polymerase chain reaction and immunofluorescence labelling.

Derivation of human embryonic stem cell lines from vitrified human embryos.

Human embryonic stem cell lines are usually derived from human embryos that have become excess to clinical needs in assisted reproduction programs, whether because the couple in question has completed their family or because the embryo was found to be clinically unsuitable for transfer due to severe genetic condition (in case of pre-implantation genetic diagnosis, PGD). Culturing embryos to a blastocyst stage (5-6 days after IVF) before embryo transfer or cryopreservation instead of earlier commonly used 8-cell stage (3 days after IVF) calls for new methods for embryo cryopreservation and allows higher efficiencies for the actual stem cell derivation. Despite the vast advances in other fields of embryonic stem cell research, methods for derivation of new lines have not changed much over the years, mainly due to scarcity of embryos limiting experimentation.

WNT and BMP signaling are both required for hematopoietic cell development from human ES cells.

Pluripotent human embryonic stem (hES) cells are capable of generating a variety of mature cell types, including hematopoietic cells in vitro. However, the precise signaling mechanisms that regulate hematopoietic cell development from hES cells are still poorly documented. Here we demonstrate that hemoangiogenic cells derived from hES cells are defined by their high-level expression of KDR and low-level expression of PDGFRalpha (KDR(+)PDGFRalpha(lo)), and that the generation of such cells from hES cells is significantly elevated by the addition of WNT3a or BMP4 during differentiation.

Thrombin-cleaved osteopontin regulates hemopoietic stem and progenitor cell functions through interactions with alpha9beta1 and alpha4beta1 integrins.

Osteopontin (OPN), a multifunctional acidic glycoprotein, expressed by osteoblasts within the endosteal region of the bone marrow (BM) suppresses the proliferation of hemopoietic stem and progenitor cells and also regulates their lodgment within the BM after transplantation. Herein we demonstrate that OPN cleavage fragments are the most abundant forms of this protein within the BM. Studies aimed to determine how hemopoietic stem cells (HSCs) interact with OPN revealed for the first time that murine and human HSCs express alpha(9)beta(1) integrin.

Investigating the interactions between haemopoietic stem cells and their niche: methods for the analysis of stem cell homing and distribution within the marrow following transplantation.

Interactions between haemopoietic stem cells (HSC) and their microenvironment serve multiple functions including the attraction to and retention and regulation in the bone marrow HSC niche. However, the cell adhesion molecules involved, their HSC receptors and the mechanisms underpinning these processes remain poorly understood. An ability to thoroughly investigate the roles of specific molecules in this process relies on a variety of in vitro and in vivo assays including the assessment of a HSC ability to home to the bone marrow and analysis of its lodgement within the bone marrow.

Endogenous fibroblastic progenitor cells in the adult mouse lung are highly enriched in the sca-1 positive cell fraction.

Originally identified as a marker specifying murine hematopoietic stem cells, the Sca-1 antigen has since been shown to be differentially expressed by candidate stem cells in tissues including vascular endothelium, skeletal muscle, mammary gland, and prostate of adult mice. In the adult murine lung, Sca-1 has previously been identified as a selectable marker for the isolation of candidate nonhematopoietic (CD45(-)), nonendothelial (CD31(-)) bronchioalveolar stem cells (BASC) located at the bronchioalveolar duct junction that coexpress surfactant protein C and the Clara cell specific protein. Our systematic analysis of CD45(-)CD31(-)Sca-1(+) cells in fetal, neonatal, and adult lung shows that very few of these cells are detectable prior to birth but expand exponentially postnatally coinciding with the transition from the saccular to the alveolar stage of lung development.

Osteopoietic stem cells: transplantable, but regeneratively limited.

Questions about the transplantability of mesenchymal stem cells, their ability to engraft within the bone marrow of recipients, and hence their clinical usefulness have been hotly debated for several decades. In this issue of Blood, Dominici and colleagues demonstrate robust serial osteopoietic engraftment, but highlight that osteopoietic chimerism declines to negligible levels after 6 months.

Tracing the life of the kidney tubule- re-establishing dogma and redirecting the options.

Renal pathology suggests that tubular repair results from tubular proliferation. In contrast, recent studies propose that postnatal kidney repair may involve renal stem cells. In this issue of Cell Stem Cell, Humphreys et al.

Neural differentiation of human embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent and capable of indefinite self-renewal in vitro. These features make them a highly advantageous source for deriving any cell type of the central and peripheral nervous system. We describe neural induction of human (h)ESCs, by using the bone morphogenic protein inhibitor protein noggin.

Innovation and stem cell excellence power - Australia's drug potential.

Australia's excellence in medical research, the transparent regulatory regime and its track record in clinical trials are driving a maturing of the country's biomedicine-dominated biotech industry. At the same time, Australia is cementing its position as an international leader in stem cell research. In terms of preclinical drug leads, Australian institutes and universities continue to generate a stream of discoveries with commercial potential.

Expansion of umbilical cord blood for clinical transplantation.

Since the first successful cord blood transplant was performed in 1988 there has been a gradual increase in the use of cord blood for hemopoietic stem cell transplantation. Worldwide, over 8,000 unrelated cord blood transplants have been performed with the majority being for children with hemopoietic malignancies. Transplantation for adults has increased but is limited by the low number of nucleated cells and CD34(+) cells within a single cord blood collection.

Gap junction mediated transport of shRNA between human embryonic stem cells.

Gap junction intracellular communication (GJIC) allows the direct transport of small molecules between adjacent cells. We hypothesized that siRNAs in one hESC could inhibit target RNA expression in another hESC via GJIC. We co-cultured green fluorescent protein (GFP)-expressing ENVY hESC with non-GFP-expressing hESC, which had been transduced to stably express shRNA directed against GFP.

The role of hyaluronic acid in hemopoietic stem cell biology.

The adult mammalian hemopoietic system maintains an extraordinarily large, yet well regulated supply of mature blood cells within the circulation throughout life. The system is capable of rapid recovery and compensation following injury, environmental stress or as a result of genetic disease such as the hemoglobinopathies. Despite the vast amount of research conducted there is still an incomplete understanding of hemopoietic regulation.

Hemopoietic stem cells with higher hemopoietic potential reside at the bone marrow endosteum.

It is now evident that hemopoietic stem cells (HSC) are located in close proximity to bone lining cells within the endosteum. Accordingly, it is unlikely that the traditional method for harvesting bone marrow (BM) from mice by simply flushing long bones would result in optimal recovery of HSC. With this in mind, we have developed improved methodologies based on sequential grinding and enzymatic digestion of murine bone tissue to harvest higher numbers of BM cells and HSC from the endosteal and central marrow regions.