Defined, feeder-free human induced pluripotent stem cell ... · # of Experiments Avg. # iPS Cell...
1
TOLL-FREE PHONE 1 800 667 0322 ∙ PHONE 1 604 877 0713 ∙ [email protected] ∙ [email protected] ∙ WWW.STEMCELL.COM ∙ FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES. Wing Y. Chang 1 , Arwen L. Hunter 1 , Erik B. Hadley 1 , Alvin Ng 1 , Jessica Norberg 1 , Jennifer Antonchuk 1 , Cynthia Fisher 3 , Terry E. Thomas 1 , Allen C. Eaves 1,2 , and Sharon A. Louis 1 1 STEMCELL Technologies Inc., Vancouver, Canada 2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, B.C., Canada 3 STEMCELL Technologies France, Grenoble, France Defined, feeder-free human induced pluripotent stem cell (hiPSC) generation, selection and expansion from multiple somatic cell types without manual colony picking or scraping Widespread adoption of human induced pluripotent stem cell (hiPSC) technology has resulted in initiatives to generate large banks of hiPSC lines. The ability to reproducibly generate hiPSCs in a high-throughput manner is hindered by challenges associated with colony identification and requirements for manual isolation of emerging colonies. Here, we show improved identification of emerging hiPSC colonies in feeder-free conditions using defined TeSR ™ -E7 ™ Medium for fibroblasts or ReproTeSR ™ Medium for peripheral blood (PB)-derived cells. Fibroblasts and blood-derived cells are the two most popular cell types for reprogramming. Further, we use a new reagent, ReLeSR ™ , for enzyme-free selective detachment of hiPSC aggregates without scraping, removal of differentiated cells or complicated manipulation. Together, these media and dissociation reagents will facilitate automation of large scale reprogramming and banking efforts. Introduction Methods 0 3 21 - 28 Day Culture Media Fibroblast Medium TeSR ™ -E7 ™ Transfect with reprogramming vectors mTeSR ™ 1 or TeSR ™ -E8 ™ Manual selection or ReLeSR ™ isolation of putative iPS colonies -7 to -10 0 3 21 - 28 Day Culture Media ReproTeSR ™ Manual selection of putative iPS colonies Isolation of PBMNCs 7 Transition to ReproTeSR ™ mTeSR ™ 1 or TeSR ™ -E8 ™ Fibroblast Reprogramming A Figure 1. Schematic of reprogramming systems with TeSR ™ -E7 ™ and ReproTeSR ™ A) Neonatal or adult fibroblasts, expanded in fibroblast medium (DMEM + 8% FBS), were transfected with episomal vectors containing OCT4, SOX2, KLF4, L-MYC and Lin28. Post-transfection (day 0), cells were seeded on Matrigel ® -coated plates at 5,000 cells/cm 2 (for later manual isolation), or 135 - 270 cells/cm 2 (density for clonal reprogramming and later ReLeSR ™ isolation). On day 3 post-transfection, media was changed to TeSR ™ -E7 ™ Medium and changed daily until day 21 - 28, when iPS colonies were identified by microscopic examination of morphology and counted. Selected colonies were transferred manually to Matrigel ® -coated plates and cultured in mTeSR ™ 1 or TeSR ™ -E8 ™ Media for further propagation of hiPSC lines. Alternatively, hiPSC colonies generated from fibroblasts seeded at a density for clonal reprogramming resulted in single colonies per well were isolated without manual selection using ReLeSR ™ and expanded in TeSR ™ -E8 ™ Medium. B, C) Mononuclear cells (MNCs) were isolated from PB by fractionation over a Ficoll ® density gradient in SepMate ™ -50 tubes. Erythroid precursor cells were expanded from PB MNCs for 7 - 10 days in StemSpan ™ SFEM II Medium with Erythroid Expansion Supplement. Alternatively, CD34 + cells were enriched from PB MNCs by immunomagnetic separation using EasySep ™ CD34 + Positive Selection Kit, and expanded for 7 days in StemSpan ™ SFEM II Medium with CD34 + Expansion Supplement. Culture expanded erythroid precursor or CD34 + cells were transfected with episomal vectors and immediately transferred to Matrigel ® -coated plates at 30,000 cells/cm 2 . Cells were transitioned from StemSpan ™ media to ReproTeSR ™ Medium by addition of 1 mL of ReproTeSR ™ on days 3 and 5. From day 7 onwards, ReproTeSR ™ reprogramming medium was replaced daily, until day 21 - 28. As above, iPS colonies were identified by microscopic examination and counted. Selected colonies were transferred manually to Matrigel ® -coated plates and cultured in mTeSR ™ 1 or TeSR ™ -E8 ™ Media for further propagation of hiPSC lines. Erythroid Cell Reprogramming B CD34 + Cell Reprogramming C Results Total Nucleated Cell Fold Expansion % CD34 + Cells After Enrichment CD34 + Cell Fold Expansion 95% CL Mean (n = 6) 39 - 74 56 12 - 25 18 15 - 22 19 Number of CD34 + Cells After Expansion per Initial 100 mL PB 0.1 - 2.5 x 10 6 1.3 x 10 6 % CD34 + Cells After Expansion 47 - 63 60 Total Number of Cells After Expansion % CD71 + /GlyA + Cells in PB MNCs % CD71 + /GlyA + Cells After Expansion 95% CL 0.23 - 1.17 0.7% 2.0 - 4.6 x 10 6 3.3 x 10 6 72 - 92 82 Number of CD71 + /GlyA + Cells After Expansion per Initial 10 mL PB 1.5 - 4.0 x 10 6 2.0 x 10 6 TABLE 1: Enrichment and expansion of CD34 + cells from human peripheral blood TABLE 2: Expansion of erythroid precursor cells from human peripheral blood Mean (n = 7) TABLE 3: Reprogramming efficiencies of fibroblasts in TeSR ™ -E7 ™ and CD34 + and erythroid precursor cells in ReproTeSR ™ Somatic Cell Type Reprogramming Efficiency (%) (mean ± SEM) Neonatal Fibroblasts (BJ line) 6 6 Avg. # iPS Cell Colonies # of Experiments 0.076 ± 0.004 0.026 ± 0.007 38 13 TeSR ™ -E7 ™ (per 50,000 input cells) PB-derived Erythroid Cells PB-derived CD34 + Cells 2 3 0.014 ± 0.003 0.012 ± 0.002 520 38 ReproTeSR ™ (per 10 mL blood) Figure 2. Superior iPSC colony morphology derived in TeSR ™ -E7 ™ or ReproTeSR ™ under feeder-free conditions. iPSC colonies were derived from fibroblasts or PB-derived CD34 + cells in defined reprogramming media TeSR ™ -E7 ™ or ReproTeSR ™ , respectively, and compared microscopically to iPSC colonies derived in hES Medium (DMEM/F12, 20% KSR, 10 ng/mL bFGF, L-glutamine, NEAA, 2-mercaptoethanol). Colonies derived in TeSR ™ -E7 ™ or ReproTeSR ™ defined media exhibited more defined borders, compact morphology, and reduced differentiation compared to those in hES Medium. TeSR ™ -E7 ™ ReproTeSR ™ Fibroblasts hES Medium hES Medium CD34 + Cells Figure 3. iPSC colonies can be selected using manual picking or ReLeSR™ selection. A) Using a pulled Pasteur pipette, selected undifferentiated areas of primary fibroblast-derived iPSC colonies were manually divided into sections of 50 - 200 μm size, and then transferred to a fresh plate for sub-cloning. B) Using ReLeSR ™ , primary iPSC colonies derived from fibroblasts seeded at a density for clonal reprogramming (135 or 270 cells/cm 2 ) were selectively detached, leaving non-reprogrammed cells attached. Aggregates of the ideal 50 - 200 μm size for sub-cloning were generated by tapping or shaking the plate after ReLeSR ™ treatment. Manual Picking ReLeSR ™ Selection Figure 4. Derivation and characterization of iPSC lines generated without manual selection using ReLeSR ™ . An iPSC colony generated at a density for clonal reprogramming was selected using ReLeSR ™ and expanded in TeSR ™ -E8 ™ Maintenance Medium. A) Colonies exhibited ideal morphology from passage 1 through passage 20; maintained high expression of markers of the undifferentiated state (OCT4 and TRA-1-60) and maintained a normal karyotype (20/20 normal cells at passage 20). B) Histological assessment of day 78 teratomas formed from passage 12 cells in the kidney or testis of SCID mice demonstrated tissues of all three germ layers. Representative tissues included: endodermal gland; mesodermal cartilage, muscle (not shown), and bone (not shown); and ectodermal pigmented cells and primitive neural cells (not shown). Scale bars = 100 μm. Adult Normal Human Dermal Fibroblasts SFEM II + Erythroid Supplement Transfect with reprogramming vectors -7 to -10 0 3 21 - 28 Day Culture Media ReproTeSR ™ Manual selection of putative iPS colonies Isolation of PBMNCs 7 Transition to ReproTeSR ™ mTeSR ™ 1 or TeSR ™ -E8 ™ SFEM II + CD34 + Supplement Transfect with reprogramming vectors “Pulled Pipette” ReLeSR ™ A Passage # 5 99.1% OCT4 + 96.4% TRA-1-60 + 20 99.9% 95.3% B Passage 1 Passage 20 20/20 Cells Normal Ectoderm: Pigmented Cells Endoderm: Gland Mesoderm: Cartilage A B Peripheral blood-derived CD34 + or erythroid cells can be efficiently expanded using StemSpan ™ SFEM II Medium with lineage-specific expansion supplements. Reprogramming in feeder-free conditions with TeSR ™ -E7 ™ (fibroblasts) and ReproTeSR ™ results in well-defined iPSC colonies which can be easily identified for selection. ReLeSR ™ can be used to select iPSC colonies for further expansion without the need for manual picking. iPSCs derived in TeSR ™ -E7 ™ or ReproTeSR ™ and selected with ReLeSR ™ are easily subcultured in mTeSR ™ 1 or TeSR ™ -E8 ™ Media to maintain a pluripotent state. Conclusions
Transcript of Defined, feeder-free human induced pluripotent stem cell ... · # of Experiments Avg. # iPS Cell...
TOLL-FREE PHONE 1 800 667 0322 ∙ PHONE 1 604 877 0713 ∙ [email protected] ∙ [email protected] ∙ WWW.STEMCELL.COM ∙ FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.
Wing Y. Chang1, Arwen L. Hunter1, Erik B. Hadley1, Alvin Ng1, Jessica Norberg1, Jennifer Antonchuk1, Cynthia Fisher3, Terry E. Thomas1, Allen C. Eaves1,2, and Sharon A. Louis1
1 STEMCELL Technologies Inc., Vancouver, Canada 2 Terry Fox Laboratory, BC Cancer Agency, Vancouver, B.C., Canada 3 STEMCELL Technologies France, Grenoble, France
Defined, feeder-free human induced pluripotent stem cell (hiPSC) generation, selection and expansion from multiple somatic cell types without manual colony picking or scraping
Widespread adoption of human induced pluripotent stem cell (hiPSC) technology has resulted in initiatives to generate large banks of hiPSC lines. The ability to reproducibly generate hiPSCs in a high-throughput manner is hindered by challenges associated with colony identification and requirements for manual isolation of emerging colonies. Here, we show improved identification of emerging hiPSC colonies in feeder-free conditions using defined TeSR™-E7™ Medium for fibroblasts or ReproTeSR™ Medium for peripheral blood (PB)-derived cells. Fibroblasts and blood-derived cells are the two most popular cell types for reprogramming. Further, we use a new reagent, ReLeSR™, for enzyme-free selective detachment of hiPSC aggregates without scraping, removal of differentiated cells or complicated manipulation. Together, these media and dissociation reagents will facilitate automation of large scale reprogramming and banking efforts.
Introduction
Methods
0 3 21 - 28Day
Culture Media Fibroblast Medium TeSR™-E7™
Transfect withreprogramming
vectors
mTeSR™1 or TeSR™-E8™
Manual selection or ReLeSR™
isolation of putative iPS colonies
-7 to -10 0 3 21 - 28Day
Culture Media ReproTeSR™
Manual selection ofputative iPS colonies
Isolationof PBMNCs
7
Transition toReproTeSR™ mTeSR™1 or TeSR™-E8™
Fibroblast ReprogrammingA
Figure 1. Schematic of reprogramming systems with TeSR™-E7™ and ReproTeSR™ A) Neonatal or adult fibroblasts, expanded in fibroblast medium (DMEM + 8% FBS), were transfected with episomal vectors containing OCT4, SOX2, KLF4, L-MYC and Lin28. Post-transfection (day 0), cells were seeded on Matrigel®-coated plates at 5,000 cells/cm2 (for later manual isolation), or 135 - 270 cells/cm2 (density for clonal reprogramming and later ReLeSR™ isolation). On day 3 post-transfection, media was changed to TeSR™-E7™ Medium and changed daily until day 21 - 28, when iPS colonies were identified by microscopic examination of morphology and counted. Selected colonies were transferred manually to Matrigel®-coated plates and cultured in mTeSR™1 or TeSR™-E8™ Media for further propagation of hiPSC lines. Alternatively, hiPSC colonies generated from fibroblasts seeded at a density for clonal reprogramming resulted in single colonies per well were isolated without manual selection using ReLeSR™ and expanded in TeSR™-E8™ Medium. B, C) Mononuclear cells (MNCs) were isolated from PB by fractionation over a Ficoll® density gradient in SepMate™-50 tubes. Erythroid precursor cells were expanded from PB MNCs for 7 - 10 days in StemSpan™ SFEM II Medium with Erythroid Expansion Supplement. Alternatively, CD34+ cells were enriched from PB MNCs by immunomagnetic separation using EasySep™ CD34+ Positive Selection Kit, and expanded for 7 days in StemSpan™ SFEM II Medium with CD34+ Expansion Supplement. Culture expanded erythroid precursor or CD34+ cells were transfected with episomal vectors and immediately transferred to Matrigel®-coated plates at 30,000 cells/cm2. Cells were transitioned from StemSpan™ media to ReproTeSR™ Medium by addition of 1 mL of ReproTeSR™ on days 3 and 5. From day 7 onwards, ReproTeSR™ reprogramming medium was replaced daily, until day 21 - 28. As above, iPS colonies were identified by microscopic examination and counted. Selected colonies were transferred manually to Matrigel®-coated plates and cultured in mTeSR™1 or TeSR™-E8™ Media for further propagation of hiPSC lines.
Erythroid Cell ReprogrammingB
CD34+ Cell ReprogrammingC
Results
Total Nucleated Cell Fold
Expansion% CD34+ Cells
After EnrichmentCD34+ Cell Fold
Expansion
95% CL
Mean(n = 6)
39 - 74
56
12 - 25
18
15 - 22
19
Number of CD34+
Cells After Expansion per Initial 100 mL PB
0.1 - 2.5 x 106
1.3 x 106
% CD34+ CellsAfter Expansion
47 - 63
60
Total Numberof Cells After
Expansion
% CD71+/GlyA+
Cells in PB MNCs% CD71+/GlyA+ Cells
After Expansion
95% CL 0.23 - 1.17
0.7%
2.0 - 4.6 x 106
3.3 x 106
72 - 92
82
Number of CD71+/GlyA+
Cells After Expansionper Initial 10 mL PB
1.5 - 4.0 x 106
2.0 x 106
TABLE 1: Enrichment and expansion of CD34+ cells from human peripheral blood
TABLE 2: Expansion of erythroid precursor cells from human peripheral blood
Mean(n = 7)
TABLE 3: Reprogramming efficiencies of fibroblasts in TeSR™-E7™ and CD34+ and erythroidprecursor cells in ReproTeSR™
Somatic Cell TypeReprogramming
Efficiency (%)(mean ± SEM)
Neonatal Fibroblasts (BJ line)
6
6
Avg. # iPS Cell Colonies# of Experiments
0.076 ± 0.004
0.026 ± 0.007
38
13
TeSR™-E7™ (per 50,000 input cells)
PB-derived Erythroid Cells
PB-derived CD34+ Cells
2
3
0.014 ± 0.003
0.012 ± 0.002
520
38
ReproTeSR™ (per 10 mL blood)
Figure 2. Superior iPSC colony morphology derived in TeSR™-E7™ or ReproTeSR™ under feeder-free conditions. iPSC colonies were derived from fibroblasts or PB-derived CD34+ cells in defined reprogramming media TeSR™-E7™ or ReproTeSR™, respectively, and compared microscopically to iPSC colonies derived in hES Medium (DMEM/F12, 20% KSR, 10 ng/mL bFGF, L-glutamine, NEAA, 2-mercaptoethanol). Colonies derived in TeSR™-E7™ or ReproTeSR™ defined media exhibited more defined borders, compact morphology, and reduced differentiation compared to those in hES Medium.
TeSR™-E7™
ReproTeSR™
Fib
rob
last
s
hES Medium
hES Medium
CD
34+ C
ells
Figure 3. iPSC colonies can be selected using manual picking or ReLeSR™ selection. A) Using a pulled Pasteur pipette, selected undifferentiated areas of primary fibroblast-derived iPSC colonies were manually divided into sections of 50 - 200 µm size, and then transferred to a fresh plate for sub-cloning. B) Using ReLeSR™, primary iPSC colonies derived from fibroblasts seeded at a density for clonal reprogramming (135 or 270 cells/cm2) were selectively detached, leaving non-reprogrammed cells attached. Aggregates of the ideal 50 - 200 µm size for sub-cloning were generated by tapping or shaking the plate after ReLeSR™ treatment.
Manual Picking
ReLeSR™ Selection
Figure 4. Derivation and characterization of iPSC lines generated without manual selection using ReLeSR™. An iPSC colony generated at a density for clonal reprogramming was selected using ReLeSR™ and expanded in TeSR™-E8™ Maintenance Medium. A) Colonies exhibited ideal morphology from passage 1 through passage 20; maintained high expression of markers of the undifferentiated state (OCT4 and TRA-1-60) and maintained a normal karyotype (20/20 normal cells at passage 20). B) Histological assessment of day 78 teratomas formed from passage 12 cells in the kidney or testis of SCID mice demonstrated tissues of all three germ layers. Representative tissues included: endodermal gland; mesodermal cartilage, muscle (not shown), and bone (not shown); and ectodermal pigmented cells and primitive neural cells (not shown). Scale bars = 100 µm.
Adult Normal Human Dermal Fibroblasts
SFEM II+ Erythroid Supplement
Transfect withreprogramming
vectors
-7 to -10 0 3 21 - 28Day
Culture Media ReproTeSR™
Manual selection ofputative iPS colonies
Isolationof PBMNCs
7
Transition toReproTeSR™ mTeSR™1 or TeSR™-E8™SFEM II
+ CD34+ Supplement
Transfect withreprogramming
vectors
“Pulled Pipette”
ReLeSR™
A
Passage #
5 99.1%
OCT4+
96.4%
TRA-1-60+
20 99.9% 95.3%
B
Passage 1
Passage 20 20/20 Cells Normal
Ectoderm: Pigmented Cells Endoderm: Gland Mesoderm: Cartilage
A
B
Peripheral blood-derived CD34+ or erythroid cells can be efficiently expanded using StemSpan™ SFEM II Medium with lineage-specific expansion supplements.
Reprogramming in feeder-free conditions with TeSR™-E7™ (fibroblasts) and ReproTeSR™ results in well-defined iPSC colonies which can be easily identified for selection.
ReLeSR™ can be used to select iPSC colonies for further expansion without the need for manual picking.
iPSCs derived in TeSR™-E7™ or ReproTeSR™ and selected with ReLeSR™ are easily subcultured in mTeSR™1 or TeSR™-E8™ Media to maintain a pluripotent state.
Conclusions
