Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Summary

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

Abstract

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.

Introduction

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.

Direct lineage reprogramming (DLR), also known as transdifferentiation, is the process of reprogramming one cell type directly into another, bypassing pluripotent and multipotent progenitor stages. DLR has thus far been used to produce numerous cell types including neural², hematopoietic^3,4,5, hepatic⁶ and nephrotic⁷, progenitor cells. For developmental biologists, DLR has become an important tool for interrogating aspects of lineage commitment and terminal differentiation processes^8,9. DLR can complement and partially replace in vivo studies for understanding mechanisms of cell fate determining factors during development. The DLR protocol for reprogramming to erythroid progenitors described in this paper provides the field a complimentary method for developmental studies of erythropoiesis.

We have previously demonstrated that overexpression of a four-factor cocktail, GATA1, TAL1, LMO2, and c-MYC (GTLM), is sufficient to reprogram both murine and human fibroblasts directly to induced erythroid progenitors (iEPs)¹⁰. The GTLM-reprogrammed erythroid cells greatly resemble bona fide primitive erythroid progenitors in terms of morphology, phenotype, and gene expression¹⁰. Thus iEPs have limited proliferation capacity and mature to nucleated erythrocytes similar to those transiently produced in the early embryo before onset of definitive erythropoiesis. By making changes in the reprogramming conditions (e.g., point mutations in reprogramming factors or addition of other factors), one can understand how this leads to changes in erythroid development and differentiation. We have for example shown that addition of Klf1 or Myb to the GTLM cocktail changes the globin expression pattern from predominantly embryonic (primitive) to mainly adult (definitive). This finding corroborates the validity of using DLR as a tool for defining developmental factors in erythropoiesis.

Here, we outline the process of generating iEPs from mouse tail tip fibroblasts (TTF). In our representative results, we performed the reprogramming on fibroblasts from the erythroid lineage-tracing mice (Epor-Cre R26-eYFP) which express the yellow fluorescent protein (eYFP) from the Rosa26 locus in all cells that have expressed the erythropoietin receptor, allowing easy visualization of commitment to the erythroid lineage. Using this method, YFP positive (EpoR+) cells are present as early as five days after transduction. This protocol, therefore, offers a quick and robust technique for the generation of erythroid progenitors in vitro.

Protocol

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 (DPBS) with 2% fetal bovine serum (FBS) until ready to use.
   NOTE: For the best results, tails should be taken from mice around 6 to 8 weeks of age. Tails can be taken from mice older than 8 weeks of age, however, as the mice age, the proliferation capability of the fibroblasts and the efficiency of reprogramming decrease¹¹.
3. Perform all subsequent steps of this protocol in a tissue culture hood under sterile conditions. Prepare a diluted trypsin solution of 0.02% trypsin-EDTA in DPBS and add 5 mL into an uncoated 10 cm dish.
4. Wash the tail, first in 70% ethanol, then in DPBS. In a dish, place the tail flat and use forceps to hold it in place. Make an incision on the tail along its longitudinal axis from the base to the tip.
5. Grasp the tail with one pair of forceps and hold it vertically. Using a second pair of forceps, grip the skin beside the incision at the base of the tail and peel it back. Do this at both sides of the incision until the skin can be peeled off by pulling downwards toward the tip of the tail.
6. Hold the peeled tail with forceps over the dish containing the trypsin solution and cut the tail into approximately 1 cm long pieces. With the tail pieces in the trypsin solution, use scissors to fragment the pieces into smaller pieces. Incubate the tail pieces in the trypsin solution at 37 °C for 10 min.
   NOTE: The smaller the pieces are preferred so as to provide each piece a high surface area to volume ratio.
7. Quench the trypsin using 2 volumes of fibroblast expansion (FEX) medium (high-glucose Dulbecco Modified Eagle Medium (DMEM) with 15% FBS, 2 mM L-glutamine, Non-essential amino acids (NEAA) and 100 U/mL Penicillin/Streptomycin).
8. Collect entire contents of the dish into a 50 mL tube and centrifuge at 350 × g for 5 min at 4 °C. Aspirate the supernatant and resuspend the tail fragments in 10 mL of fresh FEX medium.
9. Transfer tail fragments in medium to a gelatin-coated dish and incubate at 37 °C in 5% CO₂ and 4% O₂, adding fresh FEX medium every 2 days.
   NOTE: After five to seven days, tail fragments have attached at the bottom of the dish and fibroblasts can be seen moving away from them.
10. Once clusters of fibroblasts are spotted, gently shake the dish to dislodge tail pieces and aspirate the medium and all the bone fragments leaving the fibroblasts attached to the plate. Add new FEX medium and culture the fibroblasts until confluent.
11. To ensure no contamination of fibroblasts by hematopoietic progenitors, dissociate the cells from the plate using 1x trypsin-EDTA for 5 mins and collect cells. Deplete for cells expressing hematopoietic markers (CD117, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4, and Ter-119) using a magnetic separation system¹⁰.

2. Retrovirus Production

1. Seed retroviral packaging cells at approximately 2.5 × 10⁴ cells/cm² (2.0 × 10⁶ cells for a 10 cm dish) on a tissue culture-treated (by vacuum-gas plasma) dish and culture overnight in high-glucose DMEM with 10% FBS, 10 mM Sodium Pyruvate, and 100 U/mL Penicillin/Streptomycin at 37 °C and 5% CO₂.
2. On the following morning, change the medium to DMEM with no additives using half the volume used to culture overnight (5 mL for a 10 cm dish). In the afternoon, check that the cells are 70-80% confluent and begin the transfection.
3. For each reprogramming factor, Gata1, Tal1, Lmo2, and c-Myc, prepare a 2:1 mixture of the expression vector (pMX) and helper vector (containing gag and pol genes). For a 10 cm dish of fibroblasts, use 6 µg of expression vector and 3 µg of helper vector in a final volume of 100 µL in DMEM medium.
4. For each reprogramming factor, prepare 300 µL of room temperature (RT) DMEM in a sterile polystyrene tube and carefully add 27 µL of commercial transfection reagent.
   NOTE: The transfection reagent should be brought to RT before use and must be added directly into the medium as the electrostatic properties of the compound can make it stick to the plastic wall of the tube.
5. Add the plasmid mix into the transfection reagent-containing tube, vortex briefly and incubate the transfection reagent-DNA mixture for 15 min at RT. Briefly vortex the mixture and add it dropwise to the retroviral packaging cells so that the transfection reagent-DNA mix is evenly spread over the culture and incubate at 37 °C overnight.
6. 24 h after transfection, change the medium to DMEM with 20% FBS and 100 U/mL Penicillin/Streptomycin. 48 h after transfection, collect the supernatant and filter it through a 0.22 µm pore-size syringe filter.
   NOTE: Viral supernatants can be frozen to -80 °C and kept until required, although transduction is more efficient if fresh viral supernatant is used.

3. GTLM transduction and iEP harvest

1. Seed the tail tip fibroblasts at 1 × 10⁴ cells/cm² on 0.1% gelatin pre-coated dishes in FEX medium and incubate at 37 °C for 24 h.
2. The following day, prepare a transduction mixture as follows.
   1. Add 1 volume of viral supernatant for each reprogramming factor supplemented with 4 µg/mL of retroviral infection reagent (40%) to 6 volumes of FEX medium (60%).
   2. For a 10 cm dish of fibroblasts, add 1 mL of each viral supernatant (4 × viruses = 4 mL) supplemented with 4 µg/mL of retroviral infection reagent to 6 mL of FEX medium, giving a total of 10 mL transduction mixture.
3. Aspirate FEX medium from the fibroblast culture and replace it with the transduction mixture. Incubate the transduction for 4 h at 37 °C in hypoxic conditions (5% CO₂ and 4% O₂).
4. Aspirate the transduction mixture and replace it with fresh reprogramming medium (Serum-free Expansion medium (SFEM), 100 U/mLPenicillin/Streptomycin, 100 ng/mL murine Stem Cell Factor (mSCF), 10 ng/mL murine interleukin-3 (IL3), 2 U/mL human recombinant Erythropoietin (hrEPO), and 100 nM Dexamethasone).
5. Incubate for 8 daysat 37 °C in hypoxic conditions, adding fresh reprogramming medium every 2 days. After five to eight days, successful reprogramming will yield clusters of cells that have detached from the plate.
6. To collect reprogrammed cells for analysis, gently pipette up and down to harvest them directly from the dish. To harvest untransduced fibroblasts for comparison, dissociate the cells from the plate using 1x trypsin-EDTA and collect.

Results

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...

Discussion

Overexpression of a four-factor cocktail, GATA1, TAL1, LMO2, and 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 ...

Materials

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