Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling

SummaryMany signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming

Neuronal nitric oxide synthase contributes to the regulation of hematopoiesis

Nitric oxide (NO) signaling is important for the regulation of hematopoiesis. However, the role of individual NO synthase (NOS) isoforms is unclear. Our results indicate that the neuronal NOS isoform (nNOS) regulates hematopolesis in vitro and in vivo. nNOS is expressed in adult bone marrow and fetal liver and is enriched in stromal cells. There is a strong correlation between expression of nNOS in a panel of stromal cell lines established from bone marrow and fetal liver and the ability of these cell lines to support hematopoietic stem cells; furthermore, NO donor can further increase this ability. The number of colonies generated in vitro from the bone marrow and spleen of nNOS-null mutants is increased relative to wild-type or inducible- or endothelial NOS knockout mice. These results describe a new role for nNOS beyond its action in the brain and muscle and suggest a model where nNOS, expressed in stromal cells, produces NO which acts as a paracrine regulator of hematopoietic stem cells

Hematopoietic activity of a stromal cell transmembrane protein containing epidermal growth factor-like repeat motifs

Primitive hematopoietic stem cells are closely associated with discrete in vivo microenvironments. These “niches” are thought to provide the molecular signals that mediate stem cell differentiation and self-renewal. We have dissected the fetal liver microenvironment into distinct cellular components by establishing an extensive panel of stromal cell lines. One particular cell line maintains repopulating stem cells for prolonged in vitro culture periods. A subtraction cloning strategy has yielded a cDNA that encodes a cell surface glycoprotein with a restricted pattern of expression among stromal cell lines. This molecule, previously identified as delta-like/preadipocyte factor-1, contains epidermal growth factor-like repeats that are related to those in the notch/delta/serrate family of proteins. We have investigated the potential role of this molecule in hematopoietic stem/progenitor cell regulation. We show that the delta-like protein displays activity on purified stem cells by promoting the formation of “cobblestone areas” of proliferation. These cobblestone areas contain both primitive high-proliferative potential progenitors and in vivo repopulating stem cells

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation

A molecular profile of a hematopoietic stem cell niche

The hematopoietic microenvironment provides a complex molecular milieu that regulates the self-renewal and differentiation activities of stem cells. We have characterized a stem cell supportive stromal cell line, AFT024, that was derived from murine fetal liver. Highly purified in vivo transplantable mouse stem cells are maintained in AFT024 cultures at input levels, whereas other primitive progenitors are expanded. In addition, human stem cells are very effectively supported by AFT024. We suggest that the AFT024 cell line represents a component of an in vivo stem cell niche. To determine the molecular signals elaborated in this niche, we undertook a functional genomics approach that combines extensive sequence mining of a subtracted cDNA library, high-density array hybridization and in-depth bioinformatic analyses. The data have been assembled into a biological process oriented database, and represent a molecular profile of a candidate stem cell niche