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

Summary

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.

Abstract

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 – flow cytometry, confocal imaging, teratoma formation and transcriptional profiling – is also described. The newly generated lines express markers of embryonic stem cells – Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 – 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.

Introduction

The technology of induced pluripotent stem cells (iPSC) brings about potential cell replacement therapies, disease and developmental modeling, and drug and toxicological screening^1,2,3. 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 directly^4,5,<....

Protocol

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

1. Plating of amniotic fluid cells
   1. Obtain a minimum of 2.5 mL of amniotic fluid harvested in the process of amniocentesis by a physician.
      NOTE: All handling of live cells and tissue must be performed in a st....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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