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), G&#246;ran 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. Vincent's Institute of Medical Research and Department of Medicine, St Vincent's Hospital, University of Melbourne), &#193;ngel Raya (Catalan Institution for Research and Advanced Studies, Barcelona), and Vijay G. Sankaran (Broad Institute of the Massachusetts Institute of Technology and Harvard, Cambridge) for their previous contributions to this work. This work was supported by the Ragnar S&#246;derberg Foundation (to J.F.); the Swedish Research Council (to J.F.); Stiftelsen Olle Engkvist Byggm&#228;stare (to J.F.); the Swedish Foundation for Strategic Research (to J.F.); &#197;ke Wiberg's Foundation (to J.F.); a Marie Curie integration grant (to J.F.).

1. Establishment and Maintenance of Primary Mouse Tail Tip Fibroblast Cultures
<ol>
	<li>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 &#176;C. Aspirate the gelatin solution from the dish and allow it to dry for at least 2 h.</li>
	<li>Euthanize the mice by cervical dislocation. Remove the tail with scissors, cutting at the base of the tail. Put the tail in Dulbecco&#39;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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C for 10 min. 
	NOTE: The smaller the pieces are preferred so as to provide each piece a high surface area to volume ratio.</li>
	<li>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).</li>
	<li>Collect entire contents of the dish into a 50 mL tube and centrifuge at 350 &#215; g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend the tail fragments in 10 mL of fresh FEX medium.</li>
	<li>Transfer tail fragments in medium to a gelatin-coated dish and incubate at 37 &#176;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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Retrovirus Production
<ol>
	<li>Seed retroviral packaging cells at approximately 2.5 &#215; 104 cells/cm2 (2.0 &#215; 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 &#176;C and 5% CO2.</li>
	<li>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.</li>
	<li>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 &#181;g of expression vector and 3 &#181;g of helper vector in a final volume of 100 &#181;L in DMEM medium.</li>
	<li>For each reprogramming factor, prepare 300 &#181;L of room temperature (RT) DMEM in a sterile polystyrene tube and carefully add 27 &#181;L 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.</li>
	<li>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 &#176;C overnight.</li>
	<li>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 &#181;m pore-size syringe filter. 
	NOTE: Viral supernatants can be frozen to -80 &#176;C and kept until required, although transduction is more efficient if fresh viral supernatant is used.</li>
</ol>
3. GTLM transduction and iEP harvest
<ol>
	<li>Seed the tail tip fibroblasts at 1 &#215; 104 cells/cm2 on 0.1% gelatin pre-coated dishes in FEX medium and incubate at 37 &#176;C for 24 h.</li>
	<li>The following day, prepare a transduction mixture as follows.
	<ol>
		<li>Add 1 volume of viral supernatant for each reprogramming factor supplemented with 4 &#181;g/mL of retroviral infection reagent (40%) to 6 volumes of FEX medium (60%).</li>
		<li>For a 10 cm dish of fibroblasts, add 1 mL of each viral supernatant (4 &#215; viruses = 4 mL) supplemented with 4 &#181;g/mL of retroviral infection reagent to 6 mL of FEX medium, giving a total of 10 mL transduction mixture.</li>
	</ol>
	</li>
	<li>Aspirate FEX medium from the fibroblast culture and replace it with the transduction mixture. Incubate the transduction for 4 h at 37 &#176;C in hypoxic conditions (5% CO2 and 4% O2).</li>
	<li>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).</li>
	<li>Incubate for 8 daysat 37 &#176;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.</li>
	<li>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.</li>
</ol>

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The authors have no conflicts of interest to report.

Overexpression of a four-factor cocktail, GATA1,&#160;TAL1,&#160;LMO2,&#160;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 ...

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, hematopoietic3,4,5, hepatic6 and nephrotic7, progenitor cells. For developmental biologists, DLR has become an important tool for interrogating aspects of lineage commitment and terminal differentiation processes8,9. 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.

<table><tbody><tr><td>DMEM without sodium pyruvate</td><td>GE Life Sciences</td><td>SH30022.01</td><td>Culturing media for PhGP cells</td></tr><tr><td>DMEM with sodium pyruvate</td><td>GE Life Sciences</td><td>SH30243.01</td><td>Culturing media for tail tip fibroblasts</td></tr><tr><td>StemSpan Serum Free Expansion Media (SFEM)</td><td>Stem Cell Technologies</td><td>9650</td><td>Reprogramming media&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum HyClone</td><td>GE Life Sciences</td><td>SH30071.03HI</td><td>Growth factor</td></tr><tr><td>Penicillin/Streptomycin HyClone (100x)</td><td>Ge Life Sciences</td><td>SV30010</td><td>Antiboiotic</td></tr><tr><td>Non-Essential Amino Acids (100x)</td><td>Thermo Fisher</td><td>11140050</td><td>SNL media is suppplemented with this</td></tr><tr><td>Trypsin HyClone (1x)</td><td>GE Life Sciences</td><td>SH30042.01</td><td>Cell dissociation agent</td></tr><tr><td>Murine Stem Cell Factor (mSCF)</td><td>Peprotech</td><td>250-03</td><td>Added to reprogramming media</td></tr><tr><td>Recombinant Murine Il-3</td><td>Peprotech</td><td>213-13</td><td>Added to reprogramming media</td></tr><tr><td>human recombinant erythropoietin (hrEPO)</td><td>Peprotech</td><td>100-64</td><td>Added to reprogramming media</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>50-02-2&amp;nbsp;&amp;nbsp;</td><td>Added to reprogramming media</td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma</td><td>9000-70-8&amp;nbsp;</td><td>Dissovled in dH2O and used for coating plates. Ensure sterility before use.</td></tr><tr><td>Blasticidin S hydrochloride</td><td>Sigma</td><td>3/9/3513</td><td>Selection antibiotic. Affects both pro- and eukaryotic cells</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)</td><td>Ge Life Sciences</td><td>SH30850.03</td><td>Used for washing steps</td></tr><tr><td>Polybrene</td><td>Merck</td><td>TR-1003-G</td><td>Infection / Transfection&amp;nbsp; Reagent</td></tr><tr><td>FuGENE HD Transfection Reagent</td><td>Promega</td><td>E2311</td><td>Transfection Reagent for PhGP cell line</td></tr><tr><td>Millex-GP Syringe Filter Unit 0.22 &amp;micro;m</td><td>Merck</td><td>SLGP033RS</td><td>Used for filtering virus supernatant</td></tr><tr><td>BD Emerald Hypodermic Syringe</td><td>Becton Dickinson</td><td>SKU: 307736</td><td>Used for filtering virus supernatant</td></tr><tr><td>100 mm Culture Dish</td><td>Corning</td><td>430167</td><td>Cell culture</td></tr><tr><td>6-well plate</td><td>Falcon</td><td>10799541</td><td>Cell culture</td></tr><tr><td>Jeweler Forceps #5</td><td>Sklar</td><td>66-7642</td><td>Used for handling small tail fragments</td></tr><tr><td>Sklarlite Iris Scissors</td><td>Sklar</td><td>23-1149</td><td>Used for cutting the tail into small pieces</td></tr><tr><td>Lineage Cell Depletion Kit, mouse</td><td>Miltenyi Biotec</td><td>130-090-858</td><td>For depletion of hematopoietic cells in fibroblast cultures</td></tr><tr><td>CD117 MicroBeads, mouse</td><td>Miltenyi Biotec</td><td>130-091-224</td><td>For depletion of hematopoietic cells in fibroblast cultures</td></tr><tr><td>PheonixGP cells</td><td>ATCC</td><td>CRL-3215</td><td>retroviral packaging cell line</td></tr><tr><td>EcoPAC vector (pCL-Eco)&amp;nbsp;</td><td>Novus Biologicals</td><td>NBP2-29540</td><td>retroviral helper vector containing gag and pol genes</td></tr><tr><td>pMX-Gata1</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>pMX-Tal1</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>pMX-Lmo2</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>pMX-cMyc</td><td>Cloned in-lab</td><td></td><td></td></tr><tr><td>CellSens Standard 1.6 software&amp;nbsp;</td><td></td><td></td><td>Cytospin analysis software</td></tr></tbody></table>

direct lineage reprogramming of adult mouse fibroblast to erythroid progenitors

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.

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...

Watch this Scientific Journal Video about Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors at JoVE.com

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

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 ...

direct-lineage-reprogramming-adult-mouse-fibroblast-to-erythroid

Universidad de Cartagena UDEC

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Developmental Biology

6.3K Views.  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).

Video: Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Immunology and Infection

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.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Direct Reprogramming of Mouse Fibroblasts into Melanocytes

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, there is no effective long-term treatment for vitiligo. Therefore, it is imperative to develop a convenient and effective treatment for vitiligo. Regenerative medicine technology for direct reprogramming of skin cells into melanocytes seems to be a promising novel treatment of vitiligo. This involves the direct reprogramming of the patient's skin cells into functional melanocytes to help ameliorate the loss of melanocytes in patients with vitiligo. However, this method needs to be first tested on mice. Although direct reprogramming is widely used, there is no clear protocol for direct reprogramming into melanocytes. Moreover, the number of available transcription factors is overwhelming. 
Here, a concentrated lentivirus packaging system protocol is presented to produce transcription factors selected for reprogramming skin cells to melanocytes, including Sox10, Mitf, Pax3, Sox2, Sox9, and Snai2. Mouse embryonic fibroblasts (MEFs) were infected with the concentrated lentivirus for all these transcription factors for the direct reprogramming of the MEFs into induced melanocytes (iMels) in vitro. Furthermore, these transcription factors were screened, and the system was optimized for direct reprogramming to melanocytes. The expression of the characteristic markers of melanin in iMels at the gene or protein level was significantly increased. These results suggest that direct reprogramming of fibroblasts to melanocytes could be a successful new therapeutic strategy for vitiligo and confirm the mechanism of melanocyte development, which will provide the basis for further direct reprogramming of fibroblasts into melanocytes in vivo.

Here, we describe an optimized direct reprogramming system for melanocytes and a high-efficiency, concentrated virus packaging system that ensures smooth direct reprogramming.

The loss of function of melanocytes leads to vitiligo, which seriously affects the physical and mental health of the affected individuals. Presently, ...

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides fascinating prospects for the derivation of autologous transplants that circumvent histocompatibility barriers. However, progression through a pluripotent state and subsequent complete differentiation into desired lineages remains a roadblock for the clinical translation of iPSC technology because of the associated neoplastic potential and genomic instability. Recently, we and others showed that somatic cells cannot only be converted into iPSCs but also into different types of multipotent somatic stem cells by using defined factors, thereby circumventing progression through the pluripotent state. In particular, the direct conversion of human fibroblasts into induced neural progenitor cells (iNPCs) heralds the possibility of a novel autologous cell source for various applications such as cell replacement, disease modeling and drug screening. Here, we describe the isolation of adult human primary fibroblasts by skin biopsy and their efficient direct conversion into iNPCs by timely restricted expression of Oct4, Sox2, Klf4, as well as c-Myc. Sox2-positive neuroepithelial colonies appear after 17 days of induction and iNPC lines can be established efficiently by monoclonal isolation and expansion. Precise adjustment of viral multiplicity of infection and supplementation of leukemia inhibitory factor during the induction phase represent critical factors to achieve conversion efficiencies of up to 0.2%. Thus far, patient-specific iNPC lines could be expanded for more than 12 passages and uniformly display morphological and molecular features of neural stem/progenitor cells, such as the expression of Nestin and Sox2. The iNPC lines can be differentiated into neurons and astrocytes as judged by staining against TUJ1 and GFAP, respectively. In conclusion, we report a robust protocol for the derivation and direct conversion of human fibroblasts into stably expandable neural progenitor cells that might provide a cellular source for biomedical applications such as autologous neural cell replacement and disease modeling.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

Generation of&#160;induced pluripotent stem cell (iPSCs) from adult skin fibroblasts and subsequent differentiation into somatic cells provides ...

Neuroscience

Identification and Isolation of Burst-Forming Unit and Colony-Forming Unit Erythroid Progenitors from Mouse Tissue by Flow Cytometry

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming &#34;units&#34; known as BFU-e and CFU-e. Until recently, methods for the direct prospective and complete isolation of pure BFU-e and CFU-e progenitors from freshly isolated adult mouse bone marrow were not available. To address this gap, a single-cell RNA-seq (scRNAseq) dataset of mouse bone marrow was analyzed for the expression of genes coding for cell surface markers. This analysis was combined with cell fate assays, allowing the development of a novel flow cytometric approach that identifies and allows the isolation of complete and pure subsets of BFU-e and CFU-e progenitors in mouse bone marrow or spleen. This approach also identifies other progenitor subsets, including subsets enriched for basophil/mast cell and megakaryocytic potentials. The method consists of labeling fresh bone marrow or spleen cells with antibodies directed at Kit and CD55. Progenitors that express both these markers are then subdivided into five principal populations. Population 1 (P1 or CFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71med/high) contains all of the CFU-e progenitors and may be further subdivided into P1-low (CD71med CD150high) and P1-hi (CD71high CD150low), corresponding to early and late CFU-e, respectively; Population 2 (P2 or BFU-e, Kit+ CD55+ CD49fmed/low CD105med/high CD71low CD150high) contains all of the BFU-e progenitors; Population P3 (P3, Kit+ CD55+ CD49fmed/high CD105med/low CD150low CD41low) is enriched for basophil/mast cell progenitors; Population 4 (P4, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41+) is enriched for megakaryocytic progenitors; and Population 5 (P5, Kit+ CD55+ CD49fmed/high CD105med/low CD150high CD41-) contains progenitors with erythroid, basophil/mast cell, and megakaryocytic potential (EBMP) and erythroid/ megakaryocytic/ basophil-biased multipotential progenitors (MPPs). This novel approach allows greater precision when analyzing erythroid and other hematopoietic progenitors and also allows for reference to transcriptome information for each flow cytometrically defined population.

Here, we describe a novel flow cytometric method for prospective isolation of early burst-forming unit erythroid (BFU-e) and colony-forming unit erythroid (CFU-e) progenitors directly from fresh mouse bone marrow and spleen. This protocol, developed based on single-cell transcriptomic data, is the first to isolate all the tissue's erythroid progenitors with high purity.

Early erythroid progenitors were originally defined by their colony-forming potential in vitro and classified into burst-forming and colony-forming ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Biology

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...