Rebecca Pelekanos

Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

Comparison of Human Placenta- and Bone Marrow–Derived Multipotent Mesenchymal Stem Cells

Bone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs), but placenta appears to be an alternative and more readily available source. This study comprehensively compared human placenta-derived MSC (hpMSC) and human bone marrow-derived MSC (hbmMSC) in terms of cell characteristics, optimal growth conditions and in vivo safety specifically to determine if hpMSC could represent a source of human MSC for clinical trial. MSC were isolated from human placenta (hpMSC) and human bone marrow (hbmMSC) and expanded ex vivo using good manufacturing practice-compliant reagents. hpMSC and hbmMSC showed similar proliferation characteristics in different basal culture media types, fetal calf serum (FCS) concentrations, FCS heat-inactivation experiments, flask types and media replacement responsiveness. However, hpMSC and hbmMSC differed with respect to their proliferation capabilities at different seeding densities, with hbmMSC proliferating more slowly than hpMSC in every experiment. hpMSC had greater long-term growth ability than hbmMSC. MSC from both sources exhibited similar light microscopy morphology, size, cell surface phenotype, and mesodermal differentiation ability with the exception that hpMSC consistently appeared less able to differentiate to the adipogenic lineage. A comparison of both hbmMSC and hpMSC from early and medium passage cultures using single-nucleotide polymorphism (SNP) GeneChip analysis confirmed GTG-banding data that no copy number changes had been acquired during sequential passaging. In three of three informative cases (in which the gender of the delivered baby was male), hpMSC were of maternal origin. Neither hpMSC nor hbmMSC caused any acute toxicity in normal mice when injected intravenously at the same, or higher, doses than those currently used in clinical trials of hbmMSC. This study suggests that human placenta is an acceptable alternative source for human MSC and their use is currently being evaluated in clinical trials.

Mechanism of Activation of Protein Kinase JAK2 by the Growth Hormone Receptor

Introduction Class I cytokines regulate key processes such as growth, lactation, hematopoiesis, and immune function and contribute to oncogenesis. Although the extracellular domain structures of their receptors are well characterized, little is known about how the receptors activate their associated JAK (Janus kinase) protein kinases. We provide a mechanistic description for this process, focusing on the growth hormone (GH) receptor and its associated JAK2. Receptor-JAK2 activation process. (Top) Cartoons of the GH receptor basal state (state 1, left) and the active state (state 2, right) with (Bottom) transmembrane helix alignments for these states derived by modeling. GHR, GH receptor. Rationale We tested whether the receptor exists as a dimer in the inactive state by homo-FRET [fluorescence resonance energy transfer (FRET) between the proteins labeled with the same fluorophore] and other means. Then, to define receptor movements resulting from activation, we attached FRET reporters to the receptor below the cell membrane and correlated their movement with receptor activation, measured as increased cell proliferation. We controlled the position of the transmembrane helices with leucine zippers and mutagenesis, and we again monitored FRET and receptor activation. We used cysteine cross-linking data to define the faces of the transmembrane helices in contact in the basal state and verified this with molecular dynamics, which allowed us to model the activation process. We also used FRET reporters to monitor the movement of JAK2, and we matched this with molecular dynamics docking of the crystal structures of the kinase and its pseudokinase domains to derive a model for activation, which we then verified experimentally. Results We found that the GH receptor exists predominantly as a dimer in vivo, held together by its transmembrane helices. These helices are parallel in the basal state, and binding of the hormone converts them into a left-hand crossover state that induces separation of helices at the lower transmembrane boundary (hence, Box1 separation). This movement is triggered by increased proximity of the juxtamembrane sequences, a consequence of locking together of the lower module of the extracellular domain on hormone binding. This movement is triggered by increased proximity of the juxtamembrane sequences , a Both this locking and the helix state transition require rotation of the receptors, but the key outcome is separation of the Box1 sequences. Because these sequences are bound to the JAK2 FERM (4.1, ezrin, radixin, moesin) domains, this separation results in removal of the pseudokinase inhibitory domain of one JAK2, which is blocking the kinase domain of the other JAK2, and vice versa. This brings the two kinase domains into productive apposition, triggering JAK2 activation. We verified this mechanism by kinase-pseudokinase domain swap, by changes in JAK2 FRET signal on activation, by showing association of pseudokinase-kinase domain pairs, and by docking of the crystal structures. An animation of our complete model of GH receptor activation is provided at http://web-services.imb.uq.edu.au/waters/hgh.html. Conclusion The proposed mechanism will be useful in understanding the many actions of GH, which include altered growth, metabolism, and bone turnover. We expect that it may extend to other members of this important receptor family. The mechanism provides a molecular basis for understanding the oncogenic JAK2 mutations responsible for polycythemia vera and certain other hematologic disorders and may thus be of value in the design of small-molecule inhibitors of clinical applicability. Signaling from JAK (Janus kinase) protein kinases to STAT (signal transducers and activators of transcription) transcription factors is key to many aspects of biology and medicine, yet the mechanism by which cytokine receptors initiate signaling is enigmatic. We present a complete mechanistic model for activation of receptor-bound JAK2, based on an archetypal cytokine receptor, the growth hormone receptor. For this, we used fluorescence resonance energy transfer to monitor positioning of the JAK2 binding motif in the receptor dimer, substitution of the receptor extracellular domains with Jun zippers to control the position of its transmembrane (TM) helices, atomistic modeling of TM helix movements, and docking of the crystal structures of the JAK2 kinase and its inhibitory pseudokinase domain with an opposing kinase-pseudokinase domain pair. Activation of the receptor dimer induced a separation of its JAK2 binding motifs, driven by a ligand-induced transition from a parallel TM helix pair to a left-handed crossover arrangement. This separation leads to removal of the pseudokinase domain from the kinase domain of the partner JAK2 and pairing of the two kinase domains, facilitating trans-activation. This model may well generalize to other class I cytokine receptors. A molecular mechanism for transmembrane signaling by the growth hormone receptor is elucidated. [Also see Perspective by Wells and Kossiakoff] The Hormones Message The receptor for growth hormone is a well-studied representative of a family of cytokine receptors through which binding of hormone molecules at the cell surface is converted into a biochemical signal within the cell. Brooks et al. (10.1126/science.1249783; see the Perspective by Wells and Kossiakoff) used a combination of crystal structures, biophysical measurements, cell biology experiments with modified receptors, and molecular dynamics and modeling to decipher how the receptor actually transmits the information that a hormone molecule is bound. The results suggest that the receptors exist in inactive dimeric complexes in which two associated JAK2 protein kinase molecules interact in an inhibitory manner. Binding of growth hormone causes a structural change in the receptor that results in movement of the receptor intracellular domains apart from one another. This relieves the inhibition of the JAK2 molecules and allows them to activate one another, thus initiating the cellular response to the hormone.

Manufacturing of human placenta-derived mesenchymal stem cells for clinical trials.

Mesenchymal stem cells (MSC) are being used increasingly in clinical trials for a range of regenerative and inflammatory diseases. Bone marrow is the traditional source but is relatively inaccessible in large volume. MSC have now been derived from tissues other than bone marrow including placenta and adipose tissue. We have used placenta obtained after delivery as a source of MSC and have been unable to detect any marked differences from marrow‐derived MSC in terms of cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability. This report described our manufacturing process for isolating and expanding placenta‐derived human MSC and their safe infusion into the first patient in a clinical trial program of human placenta‐derived MSC.

Small Molecule Mesengenic Induction of Human Induced Pluripotent Stem Cells to Generate Mesenchymal Stem/Stromal Cells

The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single‐step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial‐like monolayer cells were generated by culturing ESCs/iPSCs in serum‐free medium containing the transforming growth factor‐β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC‐like morphology was associated with increased expression of genes, reflecting epithelial‐to‐mesenchymal transition. Both ESC‐ and iPSC‐derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N‐cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES‐MSCs and iPS‐MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES‐MSCs derived by other methods. We conclude that treatment with SB431542 in two‐dimensional cultures followed by culture‐induced epithelial‐to‐mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.

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. Of the two solid organ-specific CFU-F, only kCFU-F displayed suppression of T-cell alloreactivity in vitro, albeit to a lesser extent than bmMSC. Quantitative immunophenotyping using 81 phycoerythrin-conjugated CD antibodies demonstrated that all populations contained high percentages of cells expressing diagnostic MSC surface markers (Sca1, CD90.2, CD29, CD44), as well as others noted previously on murine MSC (CD24, CD49e, CD51, CD80, CD81, CD105). Illumina microarray expression profiling and bioinformatic analysis indicated a correlation of gene expression of 0.88-0.92 between pairwise comparisons. All populations expressed approximately 66% of genes in the pluripotency network (Plurinet), presumably reflecting their stem-like character. Furthermore, all populations expressed genes involved in immunomodulation, homing and tissue repair, suggesting these as conserved functions for MSC-like cells in solid organs. Despite this molecular congruence, strong biases in gene and protein expression and pathway activity were seen, suggesting organ-specific functions. Hence, tissue-derived MSC may also retain unique properties potentially rendering them more appropriate as cellular therapeutic agents for their organ of origin.

Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells

BackgroundFetal mesenchymal stem/stromal cells (MSC) represent a developmentally-advantageous cell type with translational potential.To enhance adult MSC migration, studies have focussed on the role of the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12), but more recent work implicates an intricate system of CXCR4 receptor dimerization, intracellular localization, multiple ligands, splice variants and nuclear accumulation. We investigated the intracellular localization of CXCR4 in fetal bone marrow-derived MSC and role of intracellular trafficking in CXCR4 surface expression and function.ResultsWe found that up to 4% of human fetal MSC have detectable surface-localized CXCR4. In the majority of cells, CXCR4 is located not at the cell surface, as would be required for ‘sensing’ migratory cues, but intracellularly. CXCR4 was identified in early endosomes, recycling endosomes, and lysosomes, indicating only a small percentage of CXCR4 travelling to the plasma membrane. Notably CXCR4 was also found in and around the nucleus, as detected with an anti-CXCR4 antibody directed specifically against CXCR4 isoform 2 differing only in N-terminal sequence. After demonstrating that endocytosis of CXCR4 is largely independent of endogenously-produced SDF-1, we next applied the cytoskeletal inhibitors blebbistatin and dynasore to inhibit endocytotic recycling. These increased the number of cells expressing surface CXCR4 by 10 and 5 fold respectively, and enhanced the number of cells migrating to SDF1 in vitro (up to 2.6 fold). These molecules had a transient effect on cell morphology and adhesion, which abated after the removal of the inhibitors, and did not alter functional stem cell properties.ConclusionsWe conclude that constitutive endocytosis is implicated in the regulation of CXCR4 membrane expression, and suggest a novel pharmacological strategy to enhance migration of systemically-transplanted cells.

Points to Consider in Designing Mesenchymal Stem Cell-Based Clinical Trials

Therapeutic applications of cells are likely to increase greatly in the future. Cell and cell-based gene therapy manufacturing facilities need to be purpose-designed and accredited by their national medicinal regulatory body. Production scientists need to work in close tandem with quality assurance and ethics committees to absolutely ensure the safety of new cellular products. In this review, we consider the need for preclinical safety and efficacy data, tissue source for manufacture of clinical grade human mesenchymal stem cells, aseptic tissue processing, indemnification, and the role of the national medicinal regulatory body in appropriate clinical trial design.

Manufacture of clinical grade human placenta-derived multipotent mesenchymal stromal cells.

Clinical grade human mesenchymal stem cells (MSC) are manufactured and used in clinical trials for a range of regenerative and inflammatory diseases. Human MSC have now been derived from tissues other than bone marrow, such as placenta, as described in this laboratory protocol. It provides instructions for clinical grade MSC manufacturing according to the Code of Good Manufacturing Practice (cGMP) principles and according to policies and procedures of our internal Quality Management System (QMS), which is based on the International Organization for Standardization (ISO) standard requirements. Relevant organizational structure and QMS elements are presented.

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.