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Stem Cell Bioengineering

Stem-Cell Bioengineering & Hematopoietic Tissue Engineering: megakaryocytic differentiation for platelet generation

The system and its importance: Megakaryocytes(Mks) are derived from hematopoietic stem cells (HSCs) and are characterized bytheir large size and rarity. Mks progress through several rounds of endomitosisto form polyploid cells with multilobated nuclei, a demarcation membrane, and aunique apoptotic program resulting in cytoplasmic extensions calledproplatelets. As the precursors to platelets, Mks are essential for bloodcoagulation and hemostasis. Yet, the molecular and cellular processes involvedin regulating and mediating Mk maturation remain poorly understood. Thus,research into megakaryopoiesis is vital to the discovery of novel approaches totreating Mk and platelet disorders such as thrombocytopenias, megakaryoblasticleukemia, and thrombocythemia. In addition, insights into the molecular basisof Mk maturation could inspire improved ex vivo culturing methods to promote Mkcommitment, expansion, and differentiation. This would enable the industrialproduction of both platelets, to be used in platelet transfusions, and matureMks and Mk progenitors for transplantation to patients to offsetthrombocytopenia associated with HSC transplants following high-dosechemotherapy. Both applications constitute challenging tissue engineeringproblems, whose solution could potentially have a profound impact ontransfusion and transplantation medicine.

The data that drive current andfuture work and the hypotheses. We carried out DNA-microarray basedgenome-scale transcriptional analyses of primary human CD34+ cells (theycontain the HSCs) differentiated ex vivo toward the Mk lineage to examine several genecategories and cellular programs, with emphasis on apoptosis and cell cycleprogression. Our data showed differential expression of several transcriptionfactors, functional apoptosis and cell-cycle related genes. Pathway analysisidentified the differential expression of several transcriptional targets of the p53 tumor suppressor protein,and we subsequently demonstrated a functional link between p53 activity and Mkpolyploidization and apoptosis. While p53 is widely known as a tumor suppressorprotein that initiates apoptosis and/or inhibits cell cycle progression inresponse to stress, p53 also appears to play a less recognized role in normalcell differentiation and development, perhaps in cooperation with p53 familymembers p63 and p73. Based on our preliminary results, the first hypothesis is that p53 partiallyregulates the cessation of endomitosis and the initiation of apoptosis duringMk maturation.

Our second hypothesisis that NFkB is also an important regulator ofMk maturation. Our transcriptional data showed that the p50 subunit of NFkB was downregulated during terminal Mkdifferentiation and that IkBa, a physiologic NFkBinhibitor, was differentially upregulated. Our preliminary data indicate that IkBa phosphorylationincreases during Mk differentiation and that NFkB activity, as measured by phosphorylated p65, decreases during late Mkdifferentiation. Our earlier work had established important roles for pO2 andpH in Mk maturation. Moreover, mechanical stress is of known physiologicalimportance in Mk disintegration and proplatelet/platelet release. p53 and NFkB orchestrate a variety of stress responses, and maythus be responsible for integrating such environmental signals affecting Mkmaturation. However, the roles of p53 and NFkBin megakaryopoiesis must be firmly established and understood prior to the investigationof their role inthese physiological responses, and potentialapplications thereof. The novelty and impact of suchhypothesized roles of p53 (and its family members p63and p73) and of NFkB on normal megakaryopoieticdifferentiation/maturation is very significant from both the applied andfundamental points of view. Thus,

Our Specific Aims are to:

  1. Establish the functional role of p53 andp53-family members (p63 and p73) in regulatingendomitosis, apoptosis, and platelet sheddingduring terminal Mk differentiation.
  2. Unravelthe molecular basis of the impact of p53 on terminal Mk differentiation byidentifying posttranslational modifications of p53 and elucidation of the p53regulon.
  3. To identify the signaling that leads to p53engagement and regulation upon stimulation of stem cells to differentiate intomegakaryocytes.4. Analyze the functional role of IkBa in Mkpolyploidization and apoptosis.

The Tools we use include RNAi-mediated down-regulation of geneexpression, high-throughput genomic assays, flow cytometry, Western analysis,DNA binding assays, and microscopic and immunohistochemical assays.