Princeton University

University of Washington

Icahn School of Medicine at Mount Sinai

Other

Ihor R Lemischka

Christoph Schaniel

Ron Weiss

Patrick Guye

Miles Miller

Ran Brosh, Sr

Kateri Moore

Ning Zheng

Patrick Paddison

Bronislaw Pytowski

Larry Witte

Priscilla E M Purnick

Laura Sheard

Carlos Filipe Pereira

Feng Li

Priscilla Purnick

Xenla Schafer

The present invention relates to a system for F-box hormone receptor regulated protein expression in mammalian cells. The system includes a silencing nucleic acid molecule comprising a first promoter and an shRNA operably linked to the first promoter, where the shRNA silences expression of a target protein. The system also includes an expression nucleic acid molecule comprising a second promoter, an F-box hormone receptor operably linked to the second promoter, and a nucleic acid molecule encoding a fusion protein comprising a degron fused to the target protein, where the nucleic acid molecule encoding the fusion protein is operably linked to the second promoter. Also disclosed are vectors comprising the system of the present application and methods of use thereof.

System for F-box hormone receptor regulated protein expression in mammalian cells

The disclosure relates to the development of methods for making hematopoietic stem cells from differentiated cells by introducing and expressing transcription factors. More particularly, the disclosure provides methods for redirecting differentiated cells to a hematopoietic stem cell state or to a hemogenic endothelial cell state by direct programming with specific combinations of transcription factors.

Methods for programming differentiated cells into hematopoietic stem cells

The present invention provides compositions and methods for programming mammalian cells to perform desired functions. In particular, the present invention provides compositions and methods for programming stem cells to differentiate into a desired cell type. A quorum sensing systems that regulates the expression of cell fate regulators is introduced into mammalian host cells, such as stem cells. The quorum sensing systems generally comprises vectors that express the components of a bacterial quorum sensing pathway, including proteins which catalyze the synthesis of an autoinducer and a gene encoding a regulatory partner of the autoinducer, and vectors in which genes encoding cell fate regulators are operably linked to a promoter induced by the autoinducer/regulatory partner complex. The system can also comprise vectors in which genes encoding additional cell fate regulators are operably linked to a promoter that is induced by a factor synthesized in response to a first stage of differentiation, so that a second stage of differentiation is triggered.

Engineered cellular pathways for programmed autoregulation of differentiation

Coronatine has been found to enhance binding of the JAZ1 degron to theF-box protein COI1, and analysis of the JAZ1 degron sequence has resulted in the identification of specific peptide sequences that bind COI1 with high affinity in the presence of coronatine. Crystal structure analysis has determined that coronatine and JA-Ile enhance the interaction between COI1 and JAZ1 via binding to a specific binding pocket on COI1. Attachment of one or more JAZ1 peptide tags as disclosed herein to a target protein in a non-plant cell expressingCOI1 or a homolog thereof results in degradation of the target protein following addition of a molecule that binds the coronatine/JA-Ile binding pocket on COI1. Therefore, provided herein are compositions, methods, and kits for targeted protein degradation.

Methods and compositions for targeted protein degradation

The present invention relates to methods and compositions for assaying embryonic stem cell maintenance. In particular, the present invention provides reporter constructs for stem cell pluripotency and differentiation and cells and organisms containing such constructs.

Embryonic stem cell self maintenance and renewal reporter

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG.(murine flk-2), FIG.(human flk-2) and FIG.(murine flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG., FIG.and FIG.; ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

Populations of cells that express flk-2 receptors

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG.(murine flk-2), FIG.(human flk-2) and FIG.(murine flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG., FIG.and FIG.ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

Populations of cells that express FLK-1 receptors

Method for isolating cells expressing flk-2

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG. (murine flk-2), FIG. (human flk-2) and FIG. (murine flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG. FIG. and FIG. ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

Soluble human flk-2 protein

Primitive hematopoietic stem cells are closely associated with discrete in vivo microenvironments. These &ldquo;niches&rdquo; 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 which encodes a cell surface glycoprotein with a restricted pattern of expression among stromal cell lines. This molecule, previously identified as dlk/Pref-1, contains EGF-like repeats which 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 dlk protein displays activity on purified stem cells by promoting the formation of &ldquo;cobblestone areas&rdquo; of proliferation. These cobblestone areas contain both primitive high-proliferative potential progenitors as well as in vivo repopulating stem cells.

Use of delta-like protein to inhibit the differentiation of stem cells

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG. 1a (murine flk-2), FIG. 1b (human flk-2) and FIG. 2 (murine flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG. 1a, FIG. 1b and FIG. 2; ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

Method for isolating stem cells expressing flk-1 receptors

Antibodies against tyrosine kinase receptor flk-1

Isolated mammalian nucleic acid molecules encoding receptor protein tyrosine kinases expressed in primitive hematopoietic cells and not expressed in mature hematopoietic cells are provided. Also included are the receptors encoded by such nucleic acid molecules; the nucleic acid molecules encoding receptor protein tyrosine kinases having the sequences shown in FIG. 1A (murine flk-2), FIG. 1B (human flk-2) and FIG. 2 (murine flk-1); the receptor protein tyrosine kinases having the amino acid sequences shown in FIG. 1A, FIG. 1B and FIG. 2; ligands for the receptors; nucleic acid sequences that encode the ligands; and methods of stimulating the proliferation and/or differentiation of primitive mammalian hematopoietic stem cells comprising contacting the stem cells with a ligand that binds to a receptor protein tyrosine kinase expressed in primitive mammalian hematopoietic cells and not expressed in mature hematopoietic cells.

Growing community of inventors

Princeton, NJ, United States of America

Ihor R Lemischka