Name: direct lineage reprogramming of adult mouse fibroblast to erythroid progenitors
Uploaded: 2018-12-14T13:00:00.000+00:00
Duration: 11 min 46 s
Description: Watch this Scientific Journal Video about Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors at JoVE.com

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 reprogramming as a tool for defining developmental factors in hematopoiesis. To support the validity of this method, iEPs can also be generated using GTLM induction of mouse embryonic fibroblasts and human foreskin fibroblasts, showing that it works for fibroblasts of different origins and across species10. In this report, we induce reprogramming of fibroblasts from the erythroid lineage-tracing mouse as a tool to visualize the conversion to an erythroid cell. Use of this mouse is useful but not critical to the protocol. We routinely perform reprogramming using multiple types of fibroblast from various mouse strains and identify iEPs by cell surface marker expression of Ter119 and CD71.
Our current method generates iEPs that exhibit a primitive erythroid phenotype as opposed to a definitive adult phenotype. One major difference between these phases of development is the expression of different globin genes. However, we have shown that addition of Klf1 and Myb to the GTLM cocktail changes iEPs&#39; globin expression pattern from predominantly embryonic to mainly adult10,14. Interestingly, addition of Gata2 and Runx1 to the four-factor cocktail and thrombopoietin in the medium biases the reprogramming process towards the megakaryocytic lineage16.
The GTLM-induced iEPs produce erythroid progenitors with a primitive gene expression signature and a limited proliferative capacity. Furthermore, the iEPs produce very few enucleated erythrocytes. The poor erythroid progenitor expansion is explained by the failure to reprogram to kit+ definitive erythroid cells, which could potentially be achieved with addition of other factors inducing direct reprogramming to iEPs with a definitive gene expression program.
Our data also suggest poor enucleation efficiency is explained by the fact GTLM-induced iEPs generate precursors with a primitive, rather than a definitive gene expression program. Several attempts at optimizing culture conditions were attempted without improved enucleation. Ongoing attempts to increase both progenitor proliferation and enucleation efficiency are therefore focused at identifying missing factors for inducing reprogramming to definitive iEPs.
For the optimal production of red iEP colonies, it is vital that cells are transduced with all four vectors and GTL genes are expressed at high levels. Unfortunately the currently most efficient method does not allow prior control of vector titers. An attempt to improve the efficiency using bicistronic lentiviral vectors was not successful, possibly due to issues with inferior stoichiometry or insufficient expression levels. While work is ongoing to improve reprogramming vectors, the most efficient method remains using combinations of freshly produced vector supernatant with the previously described retroviral vectors expressing only one factor and no selection marker gene.
GTLM reprogramming to iEPs is the only reported protocol able to produce erythroid progenitor-like cells from a committed somatic cell. As a research tool the DLR iEP method therefore has several advantages over other erythroid cell model systems such as the erythroid cell lines HiDEPs/HuDEP cells (PMID: 23533656)17. While HiDEPs/HuDEP cells are more convenient tools for large scale erythrocyte production and evaluating gene function during terminal erythropoiesis, the unique advantage of GTLM reprogramming to iEPs is the ability to directly study of the core transcriptional programs determining erythroid cell fate. GTLM reprogramming provides an invaluable platform for studying both erythropoiesis in humans and mouse, for example, to study the switch between primitive and definitive erythropoiesis. Understanding this switch is of huge interest in the potential treatment of hemoglobinopathies, such as Sickle Cell Anemia, in which researchers strive to reverse the switch in adults to fetal hemoglobin to alleviate the disease.

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 (eYFP) from the Rosa26 locus in all cells that express the erythropoietin receptor (Epor, Cre knocked into one allele of the endogenous Epor locus) transcript at any stage of their development12,13 (Figure 1A). After successful reprogramming of induced erythroid progenitors, YFP positive (EpoR+) cells are observable as early as five days after transduction. By day 5 of reprogramming, cells become round, lift from the surface of the plate and begin forming clusters. Day 5-iEPs display an erythroid precursor-like morphology, that feature a characteristic central nucleus, coarse chromatin, and blue cytoplasm after May-Gr&#252;nwald-Giemsa staining (Figure 1C). Hemoglobinization of some cells is evident by positive benzidine staining and a mildly red appearance when pelleted (Figure&#160;1D-E). In addition, a small fraction of day 5-iEPs co-express YFP and the erythroid-specific surface marker Ter119 (Figure 1F). By day 8, large YFP+ clusters can be seen. Day 8iEPs present a more differentiated erythroid phenotype than Day 5 iEPs; they are significantly smaller, have accumulated more hemoglobin, and also show significantly upregulated expression of Ter119 (Figure&#160;1B-1G). Cyto-spins of day 8-iEPs reveal erythroid-like morphology, while very few enucleated reticulocytes are observed (Figure 1H).
Gene expression analysis by quantitative polymerase chain reaction (qPCR) of bulk iEPs collected at day 8 shows that they have almost shutdown expression of fibroblast genes and have upregulated many erythroid genes including globin genes, predominantly expressing embryonic types (Figure 1I). To assess whether the reprogrammed cells transition through multipotent progenitors, we performed a time course and analyzed the expression of hematopoietic markers throughout reprogramming. We previously reported that erythroid precursor output was highest on day 6, followed by day 8, with 10.5% &#177; 4.6% and 6.6% &#177; 0.5% of live YFP+ cells co-expressing CD71 and Ter119, respectively10. There is also a population of CD41 positive cells before the appearance of Ter119 positive cells. Neither c-kit nor CD45 markers, which are usually expressed in hematopoietic progenitor and downregulated in erythropoiesis, are expressed at any point in the reprogramming (Figure 1J). These data suggest that the cells do not go through a multipotent hematopoietic progenitor or earlier stage, though do perhaps transition through a megakaryocyte-erythroid progenitor.
A reliable way to assess the efficiency of the reprogramming is to perform BFU-E colony-forming assays on the reprogrammed cells. In vitro differentiation capacity of day 5- and day 8-iEPs was assayed in methylcellulose supplemented with human erythropoietin, murine stem cell factor, and dexamethasone. After 8 days, iEPs formed two types of colonies: distinctly red (red iEP) and not-visibly red (non-red iEP). While cells from red colonies displayed erythroblast morphology, cells from non-red colonies did not resemble erythroid cells, and were irregular, had large deep blue and granular cytoplasm (Figure 2A).
Of the day 5-iEPs, approximately 1 in 1,000 formed red colonies, while only approximately 1:10,000 Day 8-iEPs formed red colonies, with a much higher ratio of non-red colonies formed (Figure 2B). This reduction in colony-forming ability between day 5- and day 8-iEPs could be explained byiEPs undergoing differentiation from days 5-8 and the GTLM expression vectors suffering silencing over time. qPCR analysis confirmed that expression of the exogenous transcript decreases significantly from days 5 to 8. As expected in successful reprogramming, expression levels of endogenous Gata1, Tal1, Lmo2, and c-Myc are induced, however, endogenous Lmo2 expression is only very lowly expressed (Figure 2C).
A point of note, and a limitation of the assay, is that non-red colonies containing macrophage-like cells outnumber the red hemoglobinized iEP colonies formed by reprogrammed fibroblasts in colony-forming assays. Colony-forming ability to create red iEP colonies can be improved by changing the stoichiometric ratios of GTLM factors. Transducing the cells with double the amount of Tal1 and Lmo2 expression vectors led to a slight increase in the ratio of red colonies over non-red colonies, while extra Gata1 led to a significant increase in the formation of red colonies and nearly a complete elimination of non-red colonies supporting the important role for these factors in erythropoiesis (Figure 2D). Interestingly, increasing the expression of c-Myc drastically increases the production of non-red colonies despite being indispensable for reprogramming. To obtain pure induced erythroid progenitors for downstream analysis, colonies can be isolated from the colony-forming assays. We performed gene expression analysis in the form of microarray on red and non-red colonies from Day 5-iEPs and colonies from 14.5 days post coitus (dpc) fetal liver (FL) and adult bone marrow (BM) for comparison. The microarray data are accessible through NCBI&#39;s Gene Expression Omnibus (GEO): GSE73344. We have previously shown that red-iEP colonies resemble the bona fide erythroid colonies (FL and BM) by gene expression10. Examination of the expression of Gata1, Tal1, Lmo2, and c-Myc in each colony shows that while the red colonies show similar levels of expression of all four factors to FL and BM, non-red colonies have lower expression of all factors, especially Gata1 (Figure 2E). To understand what the non-red colonies containing macrophage-like cells are, we surveyed the expression of cell type-specific genes in each type of colony from the microarray data. As previously reported, the red colonies express many erythroid-specific genes, similar to that of FL and BM. After revisiting the data, it is clear that the non-red colonies show high expression of a number of genes characteristic of macrophage cells14 (Figure 2F). Neither red nor non-red colonies show significantly increased expression of typical megakaryocyte genes15. Together, these data suggest that the non-red colonies more closely resemble macrophages than erythrocytes. Since non-red colonies are never observed when one of the GTLM factors are removed these macrophage-like cells seem to be formed when expression levels of one or more of the reprogramming factors Gata1, Tal1, Lmo2 are lower than required for reprogramming to iEPs. Together, this suggests that the expression levels of all four GTLM factors are important in TTF to iEP reprogramming and that heterogeneity is explained by incomplete reprogramming of individual cells, which potentially can be corrected by adjusting stoichiometric ratios.
To assess efficiency and robustness of our protocol, we tested the ability of set numbers of fibroblasts to produce visible clusters of iEP cells after 5 days when cultured in 384-well plates. We determined that out of 24 transductions of 20, 30, 40, and 50 TTFs, the number of wells with at least one iEP cluster was 3 (0.6% of plates fibroblasts), 15 (2.0% of plates fibroblasts), 15 (1.6% of plates fibroblasts), and 13 (1.1% of plates fibroblasts), respectively. This suggests that GLTM iEP reprogramming is a robust and reliable process that can reach an efficiency of 1%.
Other factors that can affect efficiency of reprogramming include the passage number of the fibroblasts and the culture conditions. We compared the reprogramming efficiency of TTFs that had been passaged three times or nine times prior to transduction. TTFs that had been passaged nine times (P9) showed a dramatic reduction in the ability to produce clusters of iEPs (Figure 3A). Interestingly, inclusion of serum in the reprogramming media completely blocks reprogramming, replacing serum with erythroid cytokines is necessary to allow the transduced factors to drive the new cell fate without compounding signals from the serum. Removal of the serum and the expression of new factors within the cell is likely to be stressful on the cells. Indeed, blocking p53 activation greatly increased the number of iEPs generated. This effect can also be seen in reprogramming of p53 knockout fibroblasts (Figure 3B-D). Inhibition of p53 signaling leads to more cell survival and better reprogramming and is one validated method for improving yield. Finally, reprogramming in hypoxic conditions is favorable but not vital for iEP production. However, transduced TTFs cultured in normoxia are much slower to reprogram and iEP clusters are observed after 10 days instead of five to eight days (Figure 3E).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58464/58464fig1.jpg" /> 
Figure 1: Forced expression of Gata1, Tal1, Lmo2, and c-Myc reprograms murine adult fibroblasts into erythroid progenitors which exhibit properties of bona fide erythroid cells. (A) Experimental design for transcription factor-mediated reprogramming of erythroid reporter (Epor-Cre R26-eYFP) tail tip fibroblasts (TTF) to EpoR+ reprogrammed cells. (B-F) Time course of iEP generation of untransduced TTFs (day 0) and bulk GTLM-transduced TTFs on days 5 and 8 (representative of n = 2-3). Transdifferentiation was evaluated by (B) live-cell, bright-field images of single wells (scale bar, 50 &#181;m); (C) May-Gr&#252;nwald&#39;s -Giemsa staining cyto-spin (scale bar, 20 &#181;m); (D) Benzidine/Giemsa staining cyto-spin (scale bar, 20 &#181;m); (E) macroscopic inspection of cell pellets; and (F) representative flow cytometry plots showing YFP/ Ter119 expression. (G) Cell diameter of iEPs harvested on days 5 and 8 measured from several cyto-spin slides, showing a decrease in cell size on day 8. Data are presented as mean &#177; SD (n = 21-25); ****p &#8804; 0.0001 by unpaired t-test.&#160;(H) Representative high-resolution benzidine/Giemsa images of GTLM-transduced TTFs on day 8. Scale bar, 5 &#181;m. (I) Relative mRNA expression of relevant fibroblast- and erythroid-specific genes including globin genes in untransduced TTFs (gray columns) versus bulk GTLM-transduced TTFs (red columns) on day 8, determined by qPCR. Data are presented as mean &#177; SD (n = 4-6 for iEPs, n = 2 for untransduced TTFs). (J)&#160;Graph showing summary of time-course flow cytometry analysis of untransduced TTF (day 0) and bulk GTLM-transduced TTF harvested at day 2, 4, 6 and 8 showing YFP, CD45, CD71 and Ter119 expression (n = 3). Data are presented as mean &#177; SD. Figure is adapted from Capellera-Garcia S, et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58464/58464fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58464/58464fig2.jpg" /> 
Figure 2: GTLM iEPs produce red and non-red colonies in BFU-E colony assays. (A) Representative bright-field and May-Gr&#252;nwald-Giemsa cytospin images of iEP-derived red and non-red colonies. Scale bars, 50 &#181;m (colony images) and 10 &#181;m (cyto-spin images).&#160;(B) Colony counts generated from plated untransduced TTFs, bulk day 5 iEPs and bulk day 8. Data are presented as mean &#177; SD (n = 3); ***p &#8804; 0.0005; ****p &#8804; 0.0001 by two-way ANOVA. (C)&#160;Relative mRNA expression of Gata1, Tal1, Lmo2, and c-Myc in untransduced TTF and bulk GTLM-transduced TTF harvested at day 5 and day 8, determined by qPCR. Primers were designed so that endogenous expression could be distinguished from total expression. Data are presented as mean &#177; SD (n = 4-6 for iEPs, n = 2 for untransduced TTF).&#160;(D) Colony counts generated from plated untransduced TTFs, and bulk day 5 iEPs generated by doubling the ratio of each of the GTLM factors. Data are presented as mean &#177; SD (n = 3); **p &#8804; 0.001; ****p &#8804; 0.0001 by two-way ANOVA.&#160;(E) Relative expression of Gata1, Tal1, Lmo2, and c-Myc in untransduced fibroblasts and picked colonies generated from GTLM-transduced iEPs (red and non-red), 14.5 dpc fetal liver and adult bone marrow, determined by microarray. Data are presented as mean &#177; SD; *p &#8804; 0.05; **p &#8804; 0.005. ***p &#8804; 0.0005; ****p &#8804; 0.0001 by two-way ANOVA.&#160;(F) Relative expression of selected genes known for their expression in erythroid (top), megakaryocyte (middle), and macrophage (bottom) cells in untransduced fibroblasts and picked colonies generated from GTLM-transduced iEPs (red and non-red), 14.5dpc fetal liver and adult bone marrow, determined by microarray. Data are presented as mean &#177; SD; *p &#8804; 0.05; **p &#8804; 0.005. ***p &#8804; 0.0005; ****p &#8804; 0.0001 by two-way ANOVA. Figure is adapted from Capellera-Garcia S, et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58464/58464fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58464/58464fig3.jpg" /> 
Figure 3: GTLM reprogramming is a robust and reliable process. (A) Graph of the number of clusters observed from 10,000 TTFs and representative pictures on day 5 of GTLM reprogramming of tail tip fibroblasts (TTF) that have undergone three passages (P3) or nine passages (P9) prior to reprogramming. Data are presented as mean &#177; SD (n = 6); scale bars are 50 &#181;m.&#160;(B-D) Representative pictures of iEP clusters 6 days after (B) GTLM reprogramming of tail tip fibroblasts; (C) GTLM reprogramming of TTFs with addition of p53DN expression vector; (D) GTLM reprogramming of p53 knockout TTFs (scale bars = 50 &#181;m). (E) Representative pictures of iEP clusters after 10 days of GTLM reprogramming performed at normoxic conditions (scale bar = 50 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58464/58464fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.3K Views.  Lund University. Directe afstamming herprogrammering van fibroblasten tot erythroïde voorvadercellen. Het is een nieuwe methode die we hebben ontwikkeld in ons lab om de processen van transcriptieregelgeving die het lot van de erythroïde cel bepalen beter te bestuderen. Het belangrijkste voordeel met het gebruik van directe aflijning herprogrammering om ontwikkeling te bestuderen is dat het een zeer eenvoudige techniek.Dus in vergelijking met bijvoorbeeld het verlies van functiestudies binnen knock-o...

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 ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Biology

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 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 ...

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 ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

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