Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Summary

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

Abstract

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide "NGG" protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

Introduction

The reprogramming of human somatic cells to the pluripotent state by overexpression of reprogramming factors has revolutionized stem cell research with applications in disease modeling, regenerative medicine, and drug development. Several non-viral reprogramming methods are available for delivery of reprogramming factors and generating iPSCs, but the process is labor intensive and not very efficient¹. The viral methods, though efficient, are associated with problems of virus integration and tumorigenicity^2,3,4. In this manuscript, we report the use of ....

Protocol

1. Reprogramming Protocol

1. Generation of human iPSC from primary human pancreatic cells
   1. Coat a 6-well plate with 1.5 mg/mL cold collagen and allow it to gel at 37 °C for 1 h.
   2. Plate early passage human primary pancreatic cells in Prigrow III medium (~1 - 1.5 × 10⁵ cells) on a collagen coated 6-well plate on day -2 to achieve approximately 2.5 × 10⁵ cells or at least 60% confluency per well on the day of transduction (day 0). For the first study, plate at least 2 - 3 wells to get one well with desired confluency on day 0. Use at least one well as a control to count the cells.
   ....

Results

In this publication, we have followed a simple but efficient protocol for the generation of iPSC from human pancreatic cells using integration or footprint-free Sendai virus vectors. Figure 1A shows a schematic representation of this reprogramming protocol. The human pancreatic cells were purchased commercially, cultured in Prigrow III medium and transduced with Sendai virus as explained above. Transduced spindle shaped pancreatic cells did not show any morph.......

Discussion

Reprogramming of human somatic cells to iPSCs has provided a major boost to the fields of basic biology research, personalized medicine, disease modeling, drug development and regenerative medicine¹⁶. Many current and widely used methods of human iPSC generation require the use of virus with the risk of integration into the host genome or episomal vectors with low reprogramming efficiency. Here, we present an efficient method for generating feeder-free iPSCs from human pancreatic cells with Sendai.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sendai viral vectors - CytoTune-iPS 2.0 Kit	Invitrogen	A16517	Thaw on ice; S No: 1
Trypsin EDTA	Gibco Life Tech	25300-054	0.05%, 100 ml; S No: 2
Rock inhibitor (Y-27632)	Milipore	SCM075	Use 10 μM; S No: 3
DMEM/F-12 medium	Invitrogen	11330-032	S No: 4
Serum replacement (KSR)	Gibco	10828028	S No: 5
DMEM	Invitrogen	11960069	1X; S No: 6
Fetal bovine serum	Thermo Scientific	SH30071.03	Aliquot; S No: 7
L-glutamine (Glutamax, 100X), liquid	Thermo Scientific	35050061	1/100; S No: 8
Non-Essential Amino Acids	Gibco	11140-050	1/100; S No: 9
2-Mercaptoethanol	Gibco	21985023	55 mM, 1/1,000; S No: 10
Hausser Hemacytometers	Hausser Scientific	02-671-54	S No: 11
0.1% Gelatin Solution	STEMCELL Technologies	7903	Incubate at 37º C for 1 hour; S No: 12
SSEA-4 antibody	Santacruz	sc-21704	1/100; S No: 13
TRA-1-81 antibody	Cell Signaling	4745S	1/200; S No: 14
OCT4 antibody	Santa Cruz	sc-5279	1/1,000; S No: 15
Collagen I, Rat Tail	Life Technologies	A10483-01	Keep cold; S No: 16
Alexa Fluor fluorescent 488/ 568 (secondary antibodies)	Invitrogen	A21202/A10042	1/2,000; S No: 17
DPBS	Hyclone	SH30028LS	1X; S No: 18
100-mm tissue culture dish	Falcon	353003	S No: 20
96-well tissue culture plate	Falcon	353078	S No: 21
6-well tissue culture plate	Falcon	353046	S No: 22
Dissecting scope	Nikon	SMZ745	S No: 23
Picking hood	NuAire	NU-301	S No: 24
15 ml Centrifuge Tube	Greiner Bio-One	188271	S No: 25
50 ml Centrifuge Tube	Greiner Bio-One	227261	S No: 26
Sodium pyruvate	Invitrogen	11360	S No: 28
β-mercaptoethanol	Sigma	M7522	S No: 29
Prigrow III medium	ABM	TM003	S No: 31
Countess™ Cell Counter	Invitrogen	C10227	S No: 32
Faxitron X-ray system	Faxitron	CellRad	S No: 33
Accutase	Innovative cell Technologies	AT-104	S No: 34
Collagenase	Life Technologies	17104019	1mg/ml stock; S No: 35
Dispase	STEMCELL Technologies	7923	S No: 36
hESC qualified matrigel	BD Biosciences	354277	To dilute, use cold DMEM/F-12; S No: 37
bFGF	R & D	233-FB	Stock 10 ug/ml; S No: 38
Paraformaldehyde	EMS	15710	4% stock in PBS; S No: 39
TRA-1-60	Santa Cruz	sc-21705	1/100; S No: 40
NANOG	ReproCELL	RCAB0004P-F	1/100; S No: 41
Tween 20	Sigma	P9416-100ML	S No: 42
Alkaline Phosphatase kit	Stemgent	00-0055	S No: 43
Cas9 protein	PNA Bio	CP01-50	Thaw and aliquot; S No: 44
Goat or donkey serum	Sigma	D9663/G9023	S No: 45
Triton X-100	Sigma	X100-100ML	S No: 46
DAPI	Thermo Scientific	D1306	S No: 47
Tris	Sigma	9285-100ML	S No: 48
NaCL	Sigma	S7653-250G	S No: 49
EDTA	Sigma	BP2482-500	S No: 50
T4 DNA ligase	NEB	M0202T	S No: 51
Mega Shortscript T7 kit	Thermo Scientific	AM1354	S No: 52
Mega Clear kit	Thermo Scientific	AM1908	S No: 53
SMC4	BD Biosciences	354357	S No: 54
Fibronectin	STEMCELL Technologies	7159	S No: 55
CloneJET cloning kit	Thermo Scientific	K1232	S No: 56
Fragment analyzerTM	Advanced Analytical		S No: 57
mTeSR1 medium kit	STEMCELL Technologies	5850	Warm to room temperature; S No: 58
Freezing medium mFreSR™	STEMCELL Technologies	5855	S No: 59
Freezing medium CryoStor®	STEMCELL Technologies	7930	S No: 60
MEFs	Globalstem	GSC-6301G	S No: 61
L-glutamine	Invitrogen	25030081	S No: 62
Human pancreatic cells	ABM	T0159	S No: 63
STEMdiff™ Neural Induction Medium	Stemcell Technologies	5835	S No: 64
RPMI	Thermofisher	11875-093	S No: 65
2% B27-insulin	Thermofisher	A1895601	S No: 66
CHIR99021	Stemcell Technologies	72052	S No: 67
IWP4	Stemcell Technologies	72552	S No: 68
2% B27	Thermofisher	17504044	S No: 69
MCDB 131	Life Technologies	10372019	S No: 70
Sodium bicarbonate	Sigma-Aldrich	S8761-100ML	S No: 71
Glucose	Sigma-Aldrich	G8270-100G	S No: 72
BSA	Proliant	68700	S No: 73
GDF8	Pepro-Tech	120-00	S No: 74
TUJ1 antibody	EMD Milipore	AB9354	S No: 75
NKX2-5 antibody	Santa Cruz	Sc-14033	S No: 76
SOX17 antibody	R & D systems	AF1924	S No: 77
Propidium iodide	Thermo Scientific	P3566	S No: 78
Amaxa 4D-nucleofector™	Lonza	AAF-1002	S No: 79
FACSAria II (cell sorter)	BD biosciences	SORP UV	S No: 80