22. Add 10 μL of 20 μM recombinant proteins to 200 μL of cells to
yield a final concentration of 1 μM.
23. The KRBH buffers should be warmed at 37
C in a water bath
before use.
24. This is a sample as low glucose. The buffer upon the cells
should be carefully collected to avoid a contamination of cells.
25. This is a sample as high glucose. The buffer upon the cells
should be carefully collected to avoid a contamination of cells.
26. This centrifugation step can remove the contaminated cells.
27. Alternative kit for mouse C-peptide is also available from
ALPCO, etc.
28. BCA assay can be also used to detect the protein concentration.
References
1. Pagliuca FW, Millman JR, Gu ¨rtler M, Segel M,
Van Dervort A, Ryu JH et al (2014) Generation of functional human pancreatic β cells
in vitro. Cell 159:428–439
2. Rezania A, Bruin JE, Arora P, Rubin A,
Batushansky I, Asadi A et al (2014) Reversal
of diabetes with insulin-producing cells derived
in vitro from human pluripotent stem cells. Nat
Biotechnol 32:1121–1133
3. Nostro MC, Sarangi F, Yang C, Holland A,
Elefanty AG, Stanley EG et al (2015) Efficient
generation of NKX6-1+ pancreatic progenitors
from multiple human pluripotent stem cell
lines. Stem Cell Rep 4:591–604
4. Agulnick AD, Ambruzs DM, Moorman MA,
Bhoumik A, Cesario RM, Payne JK et al (2015)
Insulin-producing endocrine cells differentiated in vitro from human embryonic stem
cells function in macroencapsulation devices
in vivo. Stem Cells Transl Med 4:1214–1222
5. Sakano D, Shiraki N, Kikawa K, Yamazoe T,
Kataoka M, Umeda K et al (2014) VMAT2
identified as a regulator of late-stage β-cell differentiation. Nat Chem Biol 10:141–148
6. Kaitsuka T, Noguchi H, Shiraki N, Kubo T,
Wei FY, Hakim F et al (2014) Generation of
functional insulin-producing cells from mouse
embryonic stem cells through 804G cellderived extracellular matrix and protein transduction of transcription factors. Stem Cells
Transl Med 3:114–127
7. Kaitsuka T, Kojima R, Kawabe M, Noguchi H,
Shiraki N, Kume S et al (2019) A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and
human pluripotent stem cells to insulinproducing cells. Biomed Mater. (in press)
14:045019
8. Joliot A, Prochiantz A (2004) Transduction
peptides: from technology to physiology. Nat
Cell Biol 6:189–196
9. Gump JM, Dowdy SF (2007) TAT transduction: the molecular mechanism and therapeutic
prospects. Trends Mol Med 13:443–448
10. Vive `s E, Schmidt J, Pe `legrin A (2008) Cellpenetrating and cell-targeting peptides in
drug delivery. Biochim Biophys Acta
1786:126–138
11. Matsushita M, Tomizawa K, Moriwaki A, Li
ST, Terada H, Matsui H (2001) A highefficiency protein transduction system demonstrating the role of PKA in long-lasting longterm potentiation. J Neurosci 21:6000–6007
12. Matsui H, Tomizawa K, Lu YF, Matsushita M
(2003) Protein therapy: in vivo protein transduction by polyarginine (11R) PTD and subcellular targeting delivery. Curr Protein Pept
Sci 4:151–157
13. Noguchi H, Kaneto H, Weir GC, Bonner-Weir
S (2003) PDX-1 protein containing its own
antennapedia-like protein transduction domain
can transduce pancreatic duct and islet cells.
Diabetes 52:1732–1737
14. Noguchi H, Bonner-Weir S, Wei FY,
Matsushita M, Matsumoto S (2005) BETA2/
NeuroD protein can be transduced into cells
due to an arginine- and lysine-rich sequence.
Diabetes 54:2859–2866
15. Wilson ME, Scheel D, German MS (2003)
Gene expression cascades in pancreatic development. Mech Dev 120:65–80
Pancreatic Differentiation Through Protein Transduction
95
yield a final concentration of 1 μM.
23. The KRBH buffers should be warmed at 37
C in a water bath
before use.
24. This is a sample as low glucose. The buffer upon the cells
should be carefully collected to avoid a contamination of cells.
25. This is a sample as high glucose. The buffer upon the cells
should be carefully collected to avoid a contamination of cells.
26. This centrifugation step can remove the contaminated cells.
27. Alternative kit for mouse C-peptide is also available from
ALPCO, etc.
28. BCA assay can be also used to detect the protein concentration.
References
1. Pagliuca FW, Millman JR, Gu ¨rtler M, Segel M,
Van Dervort A, Ryu JH et al (2014) Generation of functional human pancreatic β cells
in vitro. Cell 159:428–439
2. Rezania A, Bruin JE, Arora P, Rubin A,
Batushansky I, Asadi A et al (2014) Reversal
of diabetes with insulin-producing cells derived
in vitro from human pluripotent stem cells. Nat
Biotechnol 32:1121–1133
3. Nostro MC, Sarangi F, Yang C, Holland A,
Elefanty AG, Stanley EG et al (2015) Efficient
generation of NKX6-1+ pancreatic progenitors
from multiple human pluripotent stem cell
lines. Stem Cell Rep 4:591–604
4. Agulnick AD, Ambruzs DM, Moorman MA,
Bhoumik A, Cesario RM, Payne JK et al (2015)
Insulin-producing endocrine cells differentiated in vitro from human embryonic stem
cells function in macroencapsulation devices
in vivo. Stem Cells Transl Med 4:1214–1222
5. Sakano D, Shiraki N, Kikawa K, Yamazoe T,
Kataoka M, Umeda K et al (2014) VMAT2
identified as a regulator of late-stage β-cell differentiation. Nat Chem Biol 10:141–148
6. Kaitsuka T, Noguchi H, Shiraki N, Kubo T,
Wei FY, Hakim F et al (2014) Generation of
functional insulin-producing cells from mouse
embryonic stem cells through 804G cellderived extracellular matrix and protein transduction of transcription factors. Stem Cells
Transl Med 3:114–127
7. Kaitsuka T, Kojima R, Kawabe M, Noguchi H,
Shiraki N, Kume S et al (2019) A culture substratum with net-like polyamide fibers promotes the differentiation of mouse and
human pluripotent stem cells to insulinproducing cells. Biomed Mater. (in press)
14:045019
8. Joliot A, Prochiantz A (2004) Transduction
peptides: from technology to physiology. Nat
Cell Biol 6:189–196
9. Gump JM, Dowdy SF (2007) TAT transduction: the molecular mechanism and therapeutic
prospects. Trends Mol Med 13:443–448
10. Vive `s E, Schmidt J, Pe `legrin A (2008) Cellpenetrating and cell-targeting peptides in
drug delivery. Biochim Biophys Acta
1786:126–138
11. Matsushita M, Tomizawa K, Moriwaki A, Li
ST, Terada H, Matsui H (2001) A highefficiency protein transduction system demonstrating the role of PKA in long-lasting longterm potentiation. J Neurosci 21:6000–6007
12. Matsui H, Tomizawa K, Lu YF, Matsushita M
(2003) Protein therapy: in vivo protein transduction by polyarginine (11R) PTD and subcellular targeting delivery. Curr Protein Pept
Sci 4:151–157
13. Noguchi H, Kaneto H, Weir GC, Bonner-Weir
S (2003) PDX-1 protein containing its own
antennapedia-like protein transduction domain
can transduce pancreatic duct and islet cells.
Diabetes 52:1732–1737
14. Noguchi H, Bonner-Weir S, Wei FY,
Matsushita M, Matsumoto S (2005) BETA2/
NeuroD protein can be transduced into cells
due to an arginine- and lysine-rich sequence.
Diabetes 54:2859–2866
15. Wilson ME, Scheel D, German MS (2003)
Gene expression cascades in pancreatic development. Mech Dev 120:65–80
Pancreatic Differentiation Through Protein Transduction
95