Name: reprogramming primary amniotic fluid and membrane cells to pluripotency in xeno-free conditions
Uploaded: 2017-11-27T13:00:00
Description: Watch this Scientific Journal Video about Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions at JoVE.com

This work was supported by the Fonds Medizinische Forschung at the University of Zurich, Forschungskredit of the University of Zurich, The SCIEX NMSCh under Fellowships 10.216 and 12.176, The Swiss Society of Cardiology, The Swiss National Science Foundation under Grant [320030-122273] and [310030-143992], The 7th Framework Programme, Life Valve, European Commission under Grant [242008], the Olga Mayenfisch Foundation, the EMDO Foundation, the Start-up Grant 2012 of the University Hospital Zurich, and internal funding of the Mitchell Cancer Institute.

The protocol follows institutional guidelines of the ethics committee for human research. Written consent of the patient was obtained for using the amniotic fluid for research.
This protocol follows the policies of the Institutional Animal Care and Use Committee of the University of South Alabama.
1. Isolation and Culture of Primary Amniotic Mesenchymal Stem Cells
<ol>
	<li>Plating of amniotic fluid cells
	<ol>
		<li>Obtain a minimum of 2.5 mL of ...

<ol>
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Protoc. 8 (2), 223-253 (2013).</li><li>Chan, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+imaging+distinguishes+bona+fide+human+iPS+cells+from+partially+reprogrammed+cells.">Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells.</a> Nat. Biotechnol. 27 (11), 1033-1037 (2009).</li><li>Adewumi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+embryonic+stem+cell+lines+by+the+International+Stem+Cell+Initiative.">Characterization of human embryonic stem cell lines by the International Stem Cell Initiative.</a> Nat. Biotechnol. 25 (7), 803-816 (2007).</li><li>M&#252;ller, F. -. 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The initial phase of iPSC generation from fetal stem cells entails the extraction of the source cells from the fetal tissues, their culture, expansion, and introduction of the episomal reprogramming plasmids. This phase is followed by a culture period of around 14-18 days before the first fully reprogrammed colonies can be expanded. The final phase is maturation of the iPSC clones. The initial extraction of amniotic membrane stem cells is achieved by means of a combined mechanical and enzymatic digestion of the amnion. W...

The technology of induced pluripotent stem cells (iPSC) brings about potential cell replacement therapies, disease and developmental modeling, and drug and toxicological screening1,2,3. Replacement therapies can conceptually be achieved by cell injection, in-vitro differentiated tissue (such as cardiac patches) implantation, or guided regeneration by means of tissue engineering. Amniotic fluid (AFSC) and membrane stem cells (AMSC) are an excellent source of cells for these interventions either directly4,5,6,7 or as a starting cell population for reprogramming into pluripotency8,9,10,11,12.
Early approaches used undefined culture systems or reprogramming methods that require entail genomic integration of constructs9,10,11,12. A more recent study employed a xeno-free medium, even though a less defined basement membrane attachment matrix (BMM) was used, to generate iPSC from amniotic fluid epithelial cells. However, the teratoma formation assay was not included in the study along with a wealth of in-vitro and molecular data. Amniotic fluid epithelial cells were found to have a roughly 8-fold higher reprogramming efficiency when compared to neonatal fibroblasts13. In another study, mesenchymal stem cells from amniotic fluid were also found to be reprogrammed into iPSC with a much higher efficiency12.
Pluripotent stem cells can be differentiated into tissues representative of all 3 germ layers and thus have the broadest potential. Pediatric patients could benefit from the harvesting, reprogramming, and tissue engineering of their autologous amniotic fluid stem cells prenatally and amniotic membrane stem cells perinatally. Furthermore, the relatively low level of differentiation of fetal stem cells (lower than adult stem cells14,15) could theoretically aid in addressing the observed retention of epigenetic bias from source cells in iPSC16.
Here we present a protocol for reprogramming amniotic fluid and membrane stem cells to pluripotency in chemically defined xeno-free E8 medium on recombinant vitronectin17 (VTN) using episomal plasmids18. The main advantage of amniotic fluid and membrane cells as a source of cells for reprogramming lies in their availability pre- and perinatally and thus this approach would mainly benefit research into pediatric tissue engineering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	
	<tbody>
		<tr >
			<td >Tumor Dissociation Kit, human</td>
			<td >Miltenyi Biotec</td>
			<td >130-095-929</td>
			<td >tissue dissociation system, reagent kit, includes tissue dissociation tubes and tissue dissociation enzymes</td>
		</tr>
		<tr >
			<td >gentleMACS Dissociator</td>
			<td >Miltenyi Biotec</td>
			<td >130-093-235</td>
			<td >tissue dissociation system, dissociator</td>
		</tr>
		<tr >
			<td >Thermo Scientific&#8482; Shandon&#8482; Disposable Scalpel No. 10, Sterile, Individually Wrapped, 5.75 (14.6cm)</td>
			<td >Thermo-Fisher</td>
			<td >3120032</td>
			<td ></td>
		</tr>
		<tr >
			<td >70 &#181;m cell strainers</td>
			<td >Corning</td>
			<td >10054-456</td>
			<td ></td>
		</tr>
		<tr >
			<td >RPMI 1640 medium</td>
			<td >Thermo-Fisher</td>
			<td >32404014&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >rocking platform</td>
			<td >VWR</td>
			<td >40000-300</td>
			<td ></td>
		</tr>
		<tr >
			<td >50 ml centrifuge tubes</td>
			<td >Thermo-Fisher</td>
			<td >339652</td>
			<td ></td>
		</tr>
		<tr >
			<td >15 ml centrifuge tubes</td>
			<td >Thermo-Fisher</td>
			<td >339650</td>
			<td ></td>
		</tr>
		<tr >
			<td >EBM-2 basal medium</td>
			<td >Lonza</td>
			<td >CC-3156</td>
			<td >basal medium for AFMC medium</td>
		</tr>
		<tr >
			<td >FGF 2 Human (expressed in E. coli, non-glycosylated)</td>
			<td >Prospec Bio</td>
			<td >CYT-218</td>
			<td >bFGF, supplement for AFMC medium</td>
		</tr>
		<tr >
			<td >EGF Human, Pichia</td>
			<td >Prospec Bio</td>
			<td >CYT-332&#160;</td>
			<td >EGF, supplement for AFMC medium</td>
		</tr>
		<tr >
			<td >LR3 Insulin Like Growth Factor-1 Human Recombinant</td>
			<td >Prospec Bio</td>
			<td >CYT-022</td>
			<td >IGF, supplement for AFMC medium</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum, embryonic stem cell-qualified</td>
			<td >Thermo-Fisher</td>
			<td >10439024</td>
			<td >FBS</td>
		</tr>
		<tr >
			<td >Antibiotic-Antimycotic (100X)</td>
			<td >Thermo-Fisher</td>
			<td >15240062&#160;</td>
			<td >for primary AFSC/AMSC, for routine AFSC/AMSC it should not be necessary, do not use in medium for transfected cells!</td>
		</tr>
		<tr >
			<td >Accutase cell detachment solution</td>
			<td >StemCell Technologies</td>
			<td >07920</td>
			<td >cell detachment enzyme</td>
		</tr>
		<tr >
			<td >CryoStor&#8482; CS10</td>
			<td >StemCell Technologies</td>
			<td >07930</td>
			<td >complete freezing medium</td>
		</tr>
		<tr >
			<td >PBS, pH 7.4</td>
			<td >Thermo-Fisher Scientific</td>
			<td >10010023&#160;</td>
			<td ></td>
		</tr>
		<tr >
			<td >EndoFree Plasmid Maxi Kit (10)</td>
			<td >Qiagen</td>
			<td >12362</td>
			<td >for plasmid isolation</td>
		</tr>
		<tr >
			<td >pEP4 E02S EN2K</td>
			<td >Addgene</td>
			<td >20925</td>
			<td >EN2K, reprogramming factors Oct4+Sox2, Nanog+Klf4</td>
		</tr>
		<tr >
			<td >pEP4 E02S ET2K</td>
			<td >Addgene</td>
			<td >20927</td>
			<td >ET2K, reprogramming factors Oct4+Sox2, SV40LT+Klf4</td>
		</tr>
		<tr >
			<td >pCEP4-M2L</td>
			<td >Addgene</td>
			<td >20926</td>
			<td >M2L, reprogramming factors c-Myc+LIN28</td>
		</tr>
		<tr >
			<td >NanoDrop 2000c UV-Vis Spectrophotometer</td>
			<td >Thermo-Fisher</td>
			<td >ND-2000C</td>
			<td >spectrophotometer</td>
		</tr>
		<tr >
			<td >Neon&#174; Transfection System</td>
			<td >Thermo-Fisher</td>
			<td >MPK5000</td>
			<td >transfection system, components: 
			Neon pipette - transfection pipette 
			Neon device - transfection device</td>
		</tr>
		<tr >
			<td >Neon&#174; Transfection System 10 &#181;L Kit</td>
			<td >Thermo-Fisher</td>
			<td >MPK1025</td>
			<td >consumables kit for the Neon Transfection System, it contains: 
			Neon tip - transfection tip 
			Neon tube - transfection tube 
			buffer R - resuspension buffer 
			buffer E - electrolytic buffer</td>
		</tr>
		<tr >
			<td >Stemolecule&#8482; Sodium Butyrate</td>
			<td >StemGent</td>
			<td >04-0005</td>
			<td >small molecule enhancer of reprogramming</td>
		</tr>
		<tr >
			<td >TeSR-E8</td>
			<td >StemCell Technologies</td>
			<td >05940</td>
			<td >E8 medium</td>
		</tr>
		<tr >
			<td >Vitronectin XF&#8482;</td>
			<td >StemCell Technologies</td>
			<td >07180</td>
			<td >VTN, stock concentration 250 &#181;g/ml, used for coating at 1 &#181;g/cm2 in vitronectin dilution (CellAdhere) buffer</td>
		</tr>
		<tr >
			<td >CellAdhere&#8482; Dilution Buffer</td>
			<td >StemCell Technologies</td>
			<td >07183</td>
			<td >vitronectin dilution buffer</td>
		</tr>
		<tr >
			<td >UltraPure&#8482; 0.5M EDTA, pH 8.0</td>
			<td >Thermo-Fisher</td>
			<td >15575020</td>
			<td >dilute with PBS to 0.5 mM before use</td>
		</tr>
		<tr >
			<td >EVOS&#174; FL Imaging System</td>
			<td >Thermo-Fisher Scientific</td>
			<td >AMF4300</td>
			<td >LCD imaging microscope system</td>
		</tr>
		<tr >
			<td >CKX53 Inverted Microscope</td>
			<td >Olympus</td>
			<td ></td>
			<td >phase contrast cell culture microscope</td>
		</tr>
		<tr >
			<td >Pierce&#8482; 16% Formaldehyde (w/v), Methanol-free</td>
			<td >Thermo-Fisher</td>
			<td >28908</td>
			<td >dilute to 4% with PBS before use, diluted can be stored at 2-8 &#176;C for 1 week</td>
		</tr>
		<tr >
			<td >Perm Buffer III</td>
			<td >BD Biosciences</td>
			<td >558050</td>
			<td >permeabilization buffer, chill to -20 &#176;C before use</td>
		</tr>
		<tr >
			<td >Mouse IgG1, &#954; Isotype Control, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >557782</td>
			<td >isotype control for Oct3/4A, Nanog</td>
		</tr>
		<tr >
			<td >Mouse IgG1, &#954; Isotype Control, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >557783</td>
			<td >isotype control for Sox2</td>
		</tr>
		<tr >
			<td >Mouse anti-human Oct3/4 (Human Isoform A), Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >561628</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human Nanog, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >560791</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human Sox-2, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >562139</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse IgGM, &#954; Isotype Control, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >401617</td>
			<td >isotype control for TRA-1-60</td>
		</tr>
		<tr >
			<td >Mouse IgGM, &#954; Isotype Control, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >401618</td>
			<td >isotype control for TRA-1-81</td>
		</tr>
		<tr >
			<td >Mouse anti-human TRA-1-60, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >330613</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human TRA-1-81, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >330705</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse IgG1, &#954; Isotype Control, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >400129</td>
			<td >isotype control for SSEA-1</td>
		</tr>
		<tr >
			<td >Mouse IgG3, &#954; Isotype Control, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >401321</td>
			<td >isotype control for SSEA-4</td>
		</tr>
		<tr >
			<td >Mouse anti-human SSEA-1, Alexa Fluor&#174; 488</td>
			<td >BD Biosciences</td>
			<td >323010</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mouse anti-human SSEA-4, Alexa Fluor&#174; 647</td>
			<td >BD Biosciences</td>
			<td >330407</td>
			<td ></td>
		</tr>
		<tr >
			<td >Affinipure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor&#174; 488</td>
			<td >Jackson Immunoresearch</td>
			<td >115-606-068</td>
			<td >use at a dilution of 1:600 or further optimize</td>
		</tr>
		<tr >
			<td >Affinipure F(ab&#39;)2 Fragment Goat Anti-Mouse IgG+IgM, Alexa Fluor&#174; 647</td>
			<td >Jackson Immunoresearch</td>
			<td >115-546-068</td>
			<td >use at a dilution of 1:600 or further optimize</td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Thermo-Fisher Scientific</td>
			<td >D21490</td>
			<td >stock solution 10 mM, further dilute to 1:12.000 for a working solution</td>
		</tr>
		<tr >
			<td >Corning&#174; Matrigel&#174; Growth Factor Reduced, Phenol Red-Free</td>
			<td >Corning</td>
			<td >356231</td>
			<td >basement membrane matrix (BMM)</td>
		</tr>
		<tr >
			<td >scid-beige mice, female</td>
			<td >Taconic</td>
			<td >CBSCBG-F</td>
			<td ></td>
		</tr>
		<tr >
			<td >RNeasy Plus Mini Kit (50)</td>
			<td >Qiagen</td>
			<td >74134</td>
			<td >RNA isolation kit</td>
		</tr>
		<tr >
			<td >T-25 flasks, tissue culture-treated</td>
			<td >Thermo-Fisher</td>
			<td >156367</td>
			<td ></td>
		</tr>
		<tr >
			<td >T-75 flasks, tissue culture-treated</td>
			<td >Thermo-Fisher</td>
			<td >156499</td>
			<td ></td>
		</tr>
		<tr >
			<td >Nunc&#8482; tissue-culture dish</td>
			<td >Thermo-Fisher</td>
			<td >12-567-650&#160;</td>
			<td >10 cm tissue culture dish</td>
		</tr>
		<tr >
			<td >6-well plates, tissue-culture treated</td>
			<td >Thermo-Fisher</td>
			<td >140675</td>
			<td ></td>
		</tr>
		<tr >
			<td >Neubauer counting chamber (hemacytometer)</td>
			<td >VWR</td>
			<td >15170-173</td>
			<td ></td>
		</tr>
		<tr >
			<td >Mr. Frosty&#8482; Freezing Container</td>
			<td >Thermo-Fisher</td>
			<td >5100-0001&#160;</td>
			<td >freezing container</td>
		</tr>
		<tr >
			<td >FACS tubes, Round Bottom Polystyrene Test Tube, 5ml</td>
			<td >Corning</td>
			<td >352058</td>
			<td >5 ml polystyrene tubes</td>
		</tr>
		<tr >
			<td >Eppendorf tubes, 1.5 ml</td>
			<td >Thermo-Fisher</td>
			<td >05-402-96</td>
			<td >1.5 ml microcentrifuge tubes</td>
		</tr>
		<tr >
			<td >PCR tubes, 200 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >14-222-262</td>
			<td ></td>
		</tr>
		<tr >
			<td >pipette tips, 100 to 1250 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >02-707-407</td>
			<td >narrow-bore 1 mL tips</td>
		</tr>
		<tr >
			<td >pipette tips, 5 to 300 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >02-707-410</td>
			<td ></td>
		</tr>
		<tr >
			<td >pipette tips, 0.1 to 10 &#181;l</td>
			<td >Thermo-Fisher</td>
			<td >02-707-437</td>
			<td ></td>
		</tr>
		<tr >
			<td >wide-bore pipette tips, 1000 &#181;l</td>
			<td >VWR</td>
			<td >89049-166</td>
			<td >wide-bore 1 mL tips</td>
		</tr>
		<tr >
			<td >glass Pasteur pipettes</td>
			<td >Thermo-Fisher</td>
			<td >13-678-20A</td>
			<td ></td>
		</tr>
		<tr >
			<td >ethanol, 200 proof</td>
			<td >Thermo-Fisher</td>
			<td >04-355-451</td>
			<td ></td>
		</tr>
		<tr >
			<td >vortex mixer</td>
			<td >VWR</td>
			<td >10153-842</td>
			<td ></td>
		</tr>
		<tr >
			<td >chambered coverglass, 8-well, 1.5mm borosilicate glass</td>
			<td >Thermo-Fisher</td>
			<td >155409</td>
			<td >glass-bottom confocal-grade cultureware</td>
		</tr>
		<tr >
			<td >22G needles</td>
			<td >VWR</td>
			<td >82002-366</td>
			<td ></td>
		</tr>
		<tr >
			<td >insulin syringes</td>
			<td >Thermo-Fisher</td>
			<td >22-253-260</td>
			<td ></td>
		</tr>
		<tr >
			<td >Formalin solution, neutral buffered, 10%</td>
			<td >Sigma-Aldrich</td>
			<td >HT501128-4L</td>
			<td >fixation of explanted teratomas</td>
		</tr>
		<tr >
			<td >Illumina HT-12 v4 Expression BeachChip</td>
			<td >Illumina</td>
			<td >BD-103-0204</td>
			<td >expression microarray, supported by PluriTest, discontinued by manufacturer</td>
		</tr>
		<tr >
			<td >PrimeView Human Genome U219 Array Plate</td>
			<td >Thermo-Fisher</td>
			<td >901605</td>
			<td >expression microarray (formerly Affymetrix brand), soon to be supported by PluriTest</td>
		</tr>
		<tr >
			<td >GeneChip&#8482; Human Genome U133 Plus 2.0 Array</td>
			<td >Thermo-Fisher</td>
			<td >902482</td>
			<td >expression microarray (formerly Affymetrix brand), supported by CellNet, soon to be supported by PluriTest</td>
		</tr>
		<tr >
			<td >PluriTest&#174;</td>
			<td >Coriell Institute</td>
			<td ></td>
			<td >www.pluritest.org, free service for bioinformatic assessment of pluripotency, accepts microarray data - *.idat files from HT-12 v4 platform, soon to support U133, U219 microarray and RNA sequencing data</td>
		</tr>
		<tr >
			<td >CellNet</td>
			<td >Johns Hopkins University</td>
			<td ></td>
			<td >cellnet.hms.harvard.edu, free service for bioinformatic identification of cell type, including plutipotent stem cells, based on U133 microarray data - *.cel files, soon to support RNA sequencing data</td>
		</tr>
	</tbody>
</table>

reprogramming primary amniotic fluid and membrane cells to pluripotency in xeno-free conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

Informed written consent was obtained from patients before harvesting amniotic fluid for genetic testing purposes and dedicating a small aliquot of the fluid for research. No consent is required for the use of the amniotic membrane in research as the placenta represents medical waste. Amniotic fluid and membrane stem cells display typical mesenchymal properties, morphologically their cells are spindle-shaped and phase-bright. Upon reprogramming, the cells undergo mesenchymal-to-epithelial...

Watch this Scientific Journal Video about Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions at JoVE.com

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

The overall goal of this procedure is to efficiently derive induced pluripotent stem cells by means of episomal reprogramming from two different kinds of fetal stem cells. This method can help provide a source of pluripotent stem cells for tissue engineering, disease modeling, basic research, or longitudinal studies. The main advantage of this technique is that the reprogramming and the maturation of iPSCs transpires in chemically defined conditions while being available early for potential pediatric cell-based therapies.Demonstrating this procedure will be Steven McClellan, the principal investigator, and myself. Obtain placentas as soon as possible following birth. Cut nine-squared centimeter segment of the amnion, and wash it in a 50-millimeter centrifuge tube with 30 milliliters of PBS supplemented with antibiotic, antimycotic solution.Remove blood clots from the membranes, and then use a pair of fine scalpels to mince the membranes into fine pieces in a sterile 10-centimeter tissue culture dish. Use the scalpel blades to transfer the minced membrane tissue mass into one tissue dissociation tube containing 4.7 milliliters of RPMI-1640 medium. Mix in the dissociation enzymes.Mount the tube onto the tissue dissociator, and run program h_tumor_01. Then incubate the tube at 37 degrees Celsius on a rocking platform for 30 minutes. Further dilute the suspension with 35 milliliters of RPMI-1640, and apply to a 70-micron strainer placed over a 50-milliliter centrifuge tube.Centrifuge for five minutes at 200 times g at room temperature. After discarding the supernatant, resuspend the pellet in five milliliters of RPMI-1640, and count the cells using a hemocytometer. Seed the cells in cell clusters at a density of roughly 10, 000 cells per square centimeter into tissue culture flasks with freshly prepared AFMC medium supplemented with antibiotic, antimycotic solution.Harvest low passage number amniotic fluid and membrane stem cells for reprogramming from one T75 flask by adding two milliliters of cell detachment enzyme to the cell sheet. Incubate at 37 degrees Celsius for five to eight minutes. After centrifugation at 200 times g for 45 minutes, resuspend the pellet in one milliliter of PBS, and mix well to wash away serum components.Count the cells using a hemocytometer. Then adjust the cell density to 100, 000 cells per milliliter of PBS, and now quad into 1.5-milliliter microcentrifuge tubes. Place the microcentrifuge tubes in five-milliliter polystyrene tubes to allow centrifugation in a regular swing rudder, and centrifuge at 200 times g for four minutes at room temperature.After the spin, invert the tubes, and discard the supernatant into a waste container, and then perform an additional centrifugation step at 200 times g for three minutes. After the spin, use a 200-microliter pipette to carefully aspirate the remaining liquid from the tubes. Place transfection tips, tube, resuspension buffer, and electrolytic buffer into the tissue culture cabinet.Move the transfection device close so that its tube station can be placed directly into the cabinet. Fill one transfection tube with three milliliters of electrolytic buffer, and mount the tube into the station by pushing it all the way inside the slot. Take previously prepared aliquots of reprogramming plasmid solution out of the minus 80 degrees Celsius storage, and allow them to thaw at room temperature in the culture cabinet.Set the transfection parameters on the device to deliver one pulse of 40 milliseconds at 950 volts. Resuspend the pellet containing 100, 000 cells in 10 microliters of resuspension buffer. Working quickly, add 1/10 of the reprogramming plasmid solution to the resuspended cells.Mount the transfection tip onto the transfection pipette. Then aspirate the cell suspension into the transfection tip carefully avoiding formation of air bubbles. Insert the transfection pipette into the transfection tube.And press the start button on the screen at the transfection device. Wait for the screen message regarding the success of the transfection. Then remove the pipette from the tube immediately.Expel the suspension into one well of the target six-well plates. Mix in the medium from a neighboring well, and distribute the suspension equally into both wells. Repeat the transfection for each microcentrifuge tube with cells.Begin by adding 30 microliters of 0.5 millimolar EDTA, and PBS to five PCR tubes. Then load a tip onto a 10-microliter pipette set to two microliters. Hold the pipette tip at an angle at the colony edge, and carefully and gradually scrape the whole colony off the surface.Immediately aspirate the whole colony into the pipette tip, and transfer it into one of the prepared PCR tubes with EDTA. After repeating the process with four other colonies, incubate at room temperature for four to six minutes. Use a larger pipette tip to pipette each cell suspension up and down gently to break the colony down into smaller clumps.Avoid creating a single-cell suspension. Then plate the suspension directly into the target well of a recombinant vitronectin-coated 24-well plate. After repeating the process for the remaining colonies, incubate for three to six days.After four to six days, when the colonies have grown and become compact, wash the cell sheets with 300 microliters of 0.5 millimolar EDTA, and aspirate immediately. Then add another 300 microliters, and incubate at room temperature for five minutes. After aspirating the EDTA, set a one-milliliter pipette to capacity, mount to one milliliter wide bore tip, and aspirate E8 medium from the target well of a vitronectin-coated six-well plate.Squirt a stream of the medium at one well of the source plate to wash off the iPSC culture. Then transfer the suspension into the target well, and pipette up and down several times to break up the colonies into clumps of 20 to 40 cells. After plating all cells, ensure that the clumps are distributed evenly by gently rocking and shaking the plate.Then return the plate into the incubator for clump adhesion and colony growth. The amniotic fluid and membrane stem cells, which represent source cells for reprogramming display a typical mesenchymal morphology elongated and phase-bright. The cells acquire epithelial properties after they undergo the mesenchymal-to-epithelial transition, which leads to the formation of colonies with cobblestone-like cells.These colonies proliferate and create irregular cellular masses of MET cells. At the later stages of reprogramming, colonies of fully reprogrammed cells emerge, individually discernible cells with well-defined borders. These fully reprogrammed cells are present alongside MET colonies that are more numerous.A fully reprogrammed isolated mature clone is shown here. Following iPSC derivation and maturation, they can be subjected to detail characterization, and direct the differentiation into a variety of cell types. Don't forget that working with human tissues that have not been tested for the presence of pathogens can be extremely hazardous, and precautions such as personal protective equipment, and working in the biosafety cabinet should always be taken while performing this procedure.After watching this video, you should have a good understanding of how to induce pluripotency in fetal stem cells, what reagents and equipment to use, and how to routinely culture pluripotent stem cells.

reprogramming-primary-amniotic-fluid-membrane-cells-to-pluripotency

Research

JoVE Journal

Developmental Biology

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

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 ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or &#39;spheroids&#39; in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

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 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 ...