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Developmental Biology

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Published: December 14th, 2018

DOI:

10.3791/58464

1Department of Molecular Medicine and Gene Therapy, Lund University

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days.

While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Red blood cells (RBCs) are essential in all vertebrates and make up 84% of all cells of human bodies1. From embryonic to adult life, our health is highly dependent on exact regulation of RBC homeostasis. The ongoing production of mature RBCs throughout development into adulthood is known as erythropoiesis. A major challenge in erythropoiesis research is to define the master regulators that orchestrate RBC development and the switch between primitive and definitive erythropoiesis. Direct lineage reprogramming of erythroid progenitors presents an opportunity to further understand erythroid development in vivo.

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1. Establishment and Maintenance of Primary Mouse Tail Tip Fibroblast Cultures

  1. Prepare gelatin-coated dishes (recommend a 10 cm dish for one tail) by covering the surface with 0.1% gelatin and incubating the dishes for approximately 20 min at 37 °C. Aspirate the gelatin solution from the dish and allow it to dry for at least 2 h.
  2. Euthanize the mice by cervical dislocation. Remove the tail with scissors, cutting at the base of the tail. Put the tail in Dulbecco's phosphate-buffered saline (D.......

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Here we present a reproducible protocol for the production of iEPs from adult fibroblasts using transcription factor-driven DLR. We evaluate the cell reprogramming using flow cytometry, colony-forming assays, and gene expression analysis. In order to assist in the visualization of the conversion to erythroid cell fate, we performed the reprogramming on fibroblasts from the erythroid lineage-tracing mice (Epor-Cre R26-eYFP) which express the yellow fluorescent protein (eY.......

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Overexpression of a four-factor cocktail, GATA1TAL1LMO2and c-MYC (GTLM), is sufficient to reprogram murine and human fibroblasts directly to iEPs10. The reprogrammed erythroid cells greatly resembled bona fide erythroid progenitors in terms of morphology, phenotype, gene expression, and colony-forming ability. This finding corroborates the rationale of using direct .......

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We thank Evelyn Wang and Gregory Hyde (Whitehead Institute, Cambridge, MA) for cloning and Harvey Lodish (Whitehead Institute) for providing many of the plasmids used for generating the retroviral library. We thank Kavitha Siva and Sofie Singbrant (Department of Molecular Medicine and Gene Therapy, Lund University), Göran Karlsson and Shamit Soneji (Department of Molecular Hematology, Lund University) for their roles in description of iEP production. We would also like to acknowledge and thank Julian Pulecio (Centre of Regenerative Medicine, Barcelona Biomedical Research Park), Violeta Rayon-Estrada (The Rockefeller University, New York), Carl Walkley (St. Vincen....

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Name Company Catalog Number Comments
DMEM without sodium pyruvate GE Life Sciences SH30022.01 Culturing media for PhGP cells
DMEM with sodium pyruvate GE Life Sciences SH30243.01 Culturing media for tail tip fibroblasts
StemSpan Serum Free Expansion Media (SFEM) Stem Cell Technologies 9650 Reprogramming media 
Fetal Bovine Serum HyClone GE Life Sciences SH30071.03HI Growth factor
Penicillin/Streptomycin HyClone (100x) Ge Life Sciences SV30010 Antiboiotic
Non-Essential Amino Acids (100x) Thermo Fisher 11140050 SNL media is suppplemented with this
Trypsin HyClone (1x) GE Life Sciences SH30042.01 Cell dissociation agent
Murine Stem Cell Factor (mSCF) Peprotech 250-03 Added to reprogramming media
Recombinant Murine Il-3 Peprotech 213-13 Added to reprogramming media
human recombinant erythropoietin (hrEPO) Peprotech 100-64 Added to reprogramming media
Dexamethasone Sigma 50-02-2   Added to reprogramming media
Gelatin from porcine skin Sigma 9000-70-8  Dissovled in dH2O and used for coating plates. Ensure sterility before use.
Blasticidin S hydrochloride Sigma 3/9/3513 Selection antibiotic. Affects both pro- and eukaryotic cells
Dulbecco's Phosphate Buffered Saline (DPBS) Ge Life Sciences SH30850.03 Used for washing steps
Polybrene Merck TR-1003-G Infection / Transfection  Reagent
FuGENE HD Transfection Reagent Promega E2311 Transfection Reagent for PhGP cell line
Millex-GP Syringe Filter Unit 0.22 µm Merck SLGP033RS Used for filtering virus supernatant
BD Emerald Hypodermic Syringe Becton Dickinson SKU: 307736 Used for filtering virus supernatant
100 mm Culture Dish Corning 430167 Cell culture
6-well plate Falcon 10799541 Cell culture
Jeweler Forceps #5 Sklar 66-7642 Used for handling small tail fragments
Sklarlite Iris Scissors Sklar 23-1149 Used for cutting the tail into small pieces
Lineage Cell Depletion Kit, mouse Miltenyi Biotec 130-090-858 For depletion of hematopoietic cells in fibroblast cultures
CD117 MicroBeads, mouse Miltenyi Biotec 130-091-224 For depletion of hematopoietic cells in fibroblast cultures
PheonixGP cells ATCC CRL-3215 retroviral packaging cell line
EcoPAC vector (pCL-Eco)  Novus Biologicals NBP2-29540 retroviral helper vector containing gag and pol genes
pMX-Gata1 Cloned in-lab
pMX-Tal1 Cloned in-lab
pMX-Lmo2 Cloned in-lab
pMX-cMyc Cloned in-lab
CellSens Standard 1.6 software  Cytospin analysis software

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