This work was supported by the California Institute for Regenerative Medicine (RB3-02266) and the NIH (DK54441).

Notes before getting started:
<ol>
	<li>Human fetal pancreata were obtained from the Birth Defects Research Laboratory, University of Washington (Seattle, Washington, USA) who harvested the tissue. Informed consent for tissue donation, storage, and use of the samples was obtained from the donors by the center. The protocol consent statement was provided in writing and The University of California, San Diego Human Research Protections Program approved the whole study (Protocol #081237XT).</li>
	<li>It is essential to maintain both the human fetal pancreas and the container holding the pancreas on ice during the entire procedure. The dissociation and digestion should be performed as fast as possible.</li>
	<li>Send the clinic where the human fetal pancreas was obtained from the buffer for storage and transport of the pancreas. (RPMI-1640 + 10% normal human serum (NHS), 300 mM trehalose SG, penicillin/streptomycin, and fungizone).</li>
</ol>
1. Dissection of Human Fetal Pancreas
<ol>
	<li>Dissolve 5 mg of RT collagenase XI in HBSS to give a final concentration of 2.5 mg/ml. Filter sterilize the solution with a 0.22 micron syringe filter into a sterile (autoclaved) scintillation vial and store at 4 &#176;C. In a tissue culture hood, set out 2 sterile Petri dishes, a sterile small crucible (if a crucible is unavailable, a small beaker may be substituted), sterilized scissors, and forceps. Add 2 ml HBSS to one Petri dish to wash the pancreas. Aspirate liquid from the tube containing the fetal pancreas, leaving approximately 1 ml.</li>
	<li>Remove the pancreas with forceps into the 60 mm Petri dish without HBSS. For these experiments, fetal ICCs are generated from fresh pancreata with gestational ages ranging from 9 to 23 weeks. Isolation from pancreata at earlier gestionational ages is difficult due to the size of the pancreas. The protocol is likely applicable to gestational ages beyond 23 weeks, however acquisition of pancreata after this time is difficult because of federal (U.S.) restrictions. Occasionally, the fetal pancreas arrives with splenic, fat, or connective tissue attached from the original dissection. The extra tissue is easily visible to the naked eye and should be removed from the pancreas before starting the protocol.</li>
	<li>Rinse the pancreas in a minimal volume of cold HBSS (~0.5 ml) in the Petri dish.</li>
	<li>Move the cleaned pancreas to the small sterile crucible using sterile forceps and place in an ice bucket. Place the crucible in a holder for a 50 ml centrifuge tube and, using 2 pairs of scissors, vigorously slice the pancreas for several minutes. (Usually 2-5 min, but the time depends on the size and firmness of the tissue). Cutting technique is important. Hold one side of the scissors in a fixed position moving only the opposite handles. The tips of the scissors should rest on the bottom of the crucible. Continue with rapid scissor motion to break the pancreas into small pieces. The smaller the pieces of tissue, the better the collagenase will work.</li>
	<li>Add 1.5 ml of HBSS + the chopped up pancreas to the vial containing the collagenase and place in a water bath shaker set to 37 &#176;C at 200 rpm for up to 10 min. Do not check more frequently than once during the first 5 min. Digestion time depends on the tissue quality, as little as 6 min may be sufficient. The end point is when the particles are small and uniform.</li>
	<li>Fill the vial (10-15 ml) with cold HBSS to stop the collagenase activity. Place on ice and allow the clumps to settle for 10 min. Often at this point, some cell death has occurred and DNA is released into the media. This can make the media &#8216;stringy&#8217;; in this case, add 100 &#181;l DNase (10 mg/ml stock) to the solution.</li>
	<li>After the tissue in the scintillation vial has settled for 10 min, aspirate off the top layer leaving it slightly less than half full (7-8 ml). Transfer the cells to a 15 ml conical tube, add 5 ml HBSS. Centrifuge for 5 min at 300 x g.</li>
	<li>Aspirate the HBSS and resuspend the cells in 5 ml media containing RPMI-1640, glutamax, 10% normal human serum (NHS), 10 mM HEPES&#160;pH 7.2, penicillin/streptomycin, and fungizone. Plate on 60 mm Petri dish. For researchers trying to generate &#946; cells, add HGF (10 ng/ml final) to the media. Additionally, a number of groups have demonstrated that supplementing the culture media with the incretin hormone GLP-1 also increases &#946;&#160;cells28. Cells should be left in suspension for 72 hr to aggregate and form ICCs.</li>
	<li>After 72 hr in suspension, cells can be plated on the matrix of the human cell line HTB9 matrix as described previously29. Regardless of growth conditions, the media should be changed every 48 hr.</li>
</ol>
2. Immunofluorescence of Markers of Endocrine Cell Proliferation and Maturation in ICCs Grown in Suspension, Monolayer, or on Glass Coverslips
<ol>
	<li>To measure cell proliferation, BrdU labeling of either adherent cells or cells in suspension is performed for 12 hr prior to PFA fixation. BrdU reagent is diluted 1:100 and filter sterilized in RPMI-1640, glutamax, 10% NHS, 10 mM HEPES&#160;pH 7.0, penicillin/streptomycin, and fungizone.</li>
	<li>For cells in suspension: Centrifuge for 5 min at 300 x g and aspirate culture medium. Add 10 ml PBS to wash the ICCs, centrifuge for 5 min at 300 x g and aspirate. If the ICCs are going to be embedded in paraffin, please follow optional protocol 3.1-3.17. For adherent cells: Remove culture medium and wash cells with PBS as described above. Fix cells for 20 min with 4% PFA at RT, then wash twice with PBS. At this point, the plates can be wrapped with Parafilm and stored in PBS at 4 &#176;C for up to 1&#160;week.</li>
	<li>Incubate the cells with 0.2% Triton-X 100 in PBS for 10 min at RT.&#160;Wash the cells twice with PBS and apply Blocking Buffer (2% donkey serum, 2% bovine serum albumin, 50 mM glycine diluted in PBS) for 1 hr. Wash the cells with Working Buffer (Blocking Buffer diluted 1:10). Dilute the antibody in working buffer. For cells grown on glass coverslips, dilute the antibody in 60 &#956;l total volume. For cells in a 6-well dish, dilute the antibody in 600 &#181;l total volume. To stain multiple epitopes, a cocktail of antibodies may be applied. As a control, use IgG or pre immune serum in place of the specific antibody. Control samples must be incubated with control IgG from the same species as the primary antibody being used.</li>
	<li>Incubate the primary antibody either 1.5 hr at RT or O/N&#160;at 4 &#176;C. Aspirate the primary antibody and wash 3x&#160;with Working Buffer at RT.</li>
	<li>Dilute the secondary antibody at 1:200 for Rhodamine and FITC conjugated antibodies, 1:500 - 1:1,000 for the Alexa conjugated antibodies. It is important to note that all secondary antibodies are light sensitive. Cover the samples in aluminum foil to prevent loss of signal. Incubate for 1 hr at RT. 
	OPTIONAL: To visualize the nuclei, DAPI can be added at 1:500 during the secondary incubation. This is useful for quantitation of protein expression in the total cell population.</li>
	<li>Aspirate the secondary antibody and wash 3x&#160;with Working Buffer at RT. For cells grown on a coverslip, gently lift the coverslip with forceps while immersed in the buffer; wash it by immersing in PBS. For cells grown in a 6-well plate, add PBS to wash and aspirate. At this point they are ready to examine under an inverted microscope.</li>
	<li>Touch the edge of the coverslip gently on a Kimwipe to get rid of excess PBS. Add a drop of mounting gel on a glass slide and invert the coverslip onto the slide. Remove excess mounting gel by aspiration. Tap the coverslip gently with the back of a 200 &#956;l pipet tip to remove bubbles.</li>
	<li>Dry at room temperature for about 20 min or O/N at 4 &#176;C and examine under a fluorescent or confocal microscope.</li>
</ol>
3. (Optional) Immunofluorescent Staining of Proteins from Paraffin Embedded ICCs
<ol>
	<li>Prepare a 3% agarose solution and let cool to 55-60 &#176;C.</li>
	<li>Transfer the ICCs incubated in suspension to a 15 ml conical tube, and centrifuge at 1,500 x g for 5 min at room temperature.</li>
	<li>Aspirate the media from the cells and fix with 500 &#956;l 4% PFA for 10 min at RT. Do not mix the pellet, unless the pellet is very large.</li>
	<li>Wash the pellet twice with 750 &#956;l PBS. Like the 4% PFA, this waste is considered hazardous and should be disposed of according to your institution&#39;s hazardous waste specifications.</li>
	<li>Gently flick the cell pellet to loosen and add 150-200 &#956;l of the agarose down the wall of the 15 ml conical tube.</li>
	<li>Mix by gently flicking the tube, but do not form bubbles.</li>
	<li>Allow to solidify at 4 &#176;C for 5-10 min.</li>
	<li>Use a 21 G needle to dislodge the pellet and place in a fresh 15 ml conical tube.</li>
	<li>Paraffin embedding and the preparation of sections on slides can be done on site using a microtome or by your core microscopy facilities.</li>
	<li>Deparaffinize tissue, hydrate, and wash with water quickly.</li>
	<li>Add 0.2 M glycine for 30 min at RT&#160;to quench the remaining aldehyde.</li>
	<li>Wash the slide twice with PBS for 2 min each.</li>
	<li>For antigen retrieval, bring 10 mM Citrate Buffer (pH 6.0) to boil over hot plate. Immerse the slides in the buffer, wait for it to come back to a boil, and wait for 15 min.</li>
	<li>Remove the beaker from the hotplate. Wait approximately 40 min&#160;for the slides to cool in the buffer.</li>
	<li>Wash the slides twice with H2O for 5 min each, wash the slides twice with PBS for 5 min each, block with 2% normal donkey serum in PBS for 1 hr.</li>
	<li>Incubate with primary antibody, diluted to correct concentration in Working Buffer containing 0.2% Triton X-100. Generally, the primary antibody is incubated overnight&#160;if staining for transcription factors and for 1.5 hr if staining for hormones, markers of proliferation, and/or differentiation.</li>
	<li>Follow steps 2.3-2.9&#160;described above.</li>
</ol>

<ol>
	<li>Tuch, B. E., Ng, A. B. P., Jones, A., Turtle, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+differentiation+of+human+fetal+pancreatic+explants+transplanted+into+nude+mice.">Histologic differentiation of human fetal pancreatic explants transplanted into nude mice.</a> Diabetes. 33, 1180-1187 (1984).</li><li>Tuch, B. E., Grigoriou, S., Turtle, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+hormonal+content+of+human+fetal+pancreas+passaged+in+athymic+mice.">Growth and hormonal content of human fetal pancreas passaged in athymic mice.</a> Diabetes. 35, 464-469 (1986).</li><li>Hullett, D. A., Falany, J. L., Love, R. B., Butler, J. A., Sollinger, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+pancreas:+Potential+for+transplantation.">Human fetal pancreas: Potential for transplantation.</a> Transplantation Proceedings. XIX (1), 909-910 (1987).</li><li>Hullett, D. A., Bethke, K. P., Landry, A. S., Leonard, D. K., Sollinger, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+long-term+cryopreservation+and+transplantation+of+human+fetal+pancreas.">Successful long-term cryopreservation and transplantation of human fetal pancreas.</a> Diabetes. 38, 448-453 (1989).</li><li>Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+diabetes+by+human+fetal+pancreas.">Reversal of diabetes by human fetal pancreas.</a> Transplantation. 51, 557-562 (1991).</li><li>Beattie, G. M., Lopez, A. D., Otonkoski, T., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+human+fetal+pancreas:+fresh+vs.+cultured+fetal+islets+or+ICCS.">Transplantation of human fetal pancreas: fresh vs. cultured fetal islets or ICCS.</a> J Mol Med. 77, 70-73 (1999).</li><li>Joglekar, M. V., 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=The+miR-30+family+microRNAs+confer+epithelial+phenotype+to+human+pancreatic+cells.">The miR-30 family microRNAs confer epithelial phenotype to human pancreatic cells.</a> Islets. 1, 137-147 (2009).</li><li>Joglekar, M. V., Joglekar, V. M., Joglekar, S. V., Hardikar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+fetal+pancreatic+insulin-producing+cells+proliferate+in+vitro.">Human fetal pancreatic insulin-producing cells proliferate in vitro.</a> J Endocrinol. 201, 27-36 (2009).</li><li>Kayali, A. G., 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=The+SDF-1alpha/CXCR4+Axis+is+Required+for+Proliferation+and+Maturation+of+Human+Fetal+Pancreatic+Endocrine+Progenitor+Cells.">The SDF-1alpha/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells.</a> PLoS One. 7, (2012).</li><li>Otonkoski, T., 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=Hepatocyte+growth+factor/scatter+factor+has+insulinotropic+activity+in+human+fetal+pancreatic+cells.">Hepatocyte growth factor/scatter factor has insulinotropic activity in human fetal pancreatic cells.</a> Diabetes. 43, 947-953 (1994).</li><li>D'Amour, K. A., 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=Production+of+pancreatic+hormone-expressing+endocrine+cells+from+human+embryonic+stem+cells.">Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells.</a> Nat Biotechnol. 24, 1392-1401 (2006).</li><li>Xu, X., 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=Endoderm+and+pancreatic+islet+lineage+differentiation+from+human+embryonic+stem+cells.">Endoderm and pancreatic islet lineage differentiation from human embryonic stem cells.</a> Cloning Stem Cells. 8, 96-107 (2006).</li><li>Jiang, J., 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=Generation+of+insulin-producing+islet-like+clusters+from+human+embryonic+stem+cells.">Generation of insulin-producing islet-like clusters from human embryonic stem cells.</a> Stem Cells. 25, 1940-1953 (2007).</li><li>Shim, J. H., 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=Directed+differentiation+of+human+embryonic+stem+cells+towards+a+pancreatic+cell+fate.">Directed differentiation of human embryonic stem cells towards a pancreatic cell fate.</a> Diabetologia. 50, 1228-1238 (2007).</li><li>Kroon, E., 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=Pancreatic+endoderm+derived+from+human+embryonic+stem+cells+generates+glucose-responsive+insulin-secreting+cells+in+vivo.">Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo.</a> Nat Biotechnol. 26, 443-452 (2008).</li><li>Van Hoof, D., D'Amour, K. A., German, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+insulin-producing+cells+from+human+embryonic+stem+cells.">Derivation of insulin-producing cells from human embryonic stem cells.</a> Stem Cell Res. 3, 73-87 (2009).</li><li>Schulz, T. C., 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=A+scalable+system+for+production+of+functional+pancreatic+progenitors+from+human+embryonic+stem+cells.">A scalable system for production of functional pancreatic progenitors from human embryonic stem cells.</a> PLoS One. 7, (2012).</li><li>Lees, J. G., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+embryonic+stem+cells+into+pancreatic+beta-cell+surrogates+guided+by+ontogeny.">Conversion of embryonic stem cells into pancreatic beta-cell surrogates guided by ontogeny.</a> Regen Med. 1, 327-336 (2006).</li><li>Rezania, A., 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=Maturation+of+human+embryonic+stem+cell-derived+pancreatic+progenitors+into+functional+islets+capable+of+treating+pre-existing+diabetes+in+mice.">Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice.</a> Diabetes. 61, 2016-2029 (2012).</li><li>Bai, L., Meredith, G., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon-like+peptide-1+enhances+production+of+insulin+in+insulin-producing+cells+derived+from+mouse+embryonic+stem+cells.">Glucagon-like peptide-1 enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells.</a> J Endocrinol. 186, 343-352 (2005).</li><li>Semb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+endoderm:+a+key+step+in+coaxing+human+embryonic+stem+cells+into+transplantable+beta-cells.">Definitive endoderm: a key step in coaxing human embryonic stem cells into transplantable beta-cells.</a> Biochem Soc Trans. 36, 272-275 (2008).</li><li>Van Hoof, D., Mendelsohn, A. D., Seerke, R., Desai, T. A., German, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+into+pancreatic+endoderm+in+patterned+size-controlled+clusters.">Differentiation of human embryonic stem cells into pancreatic endoderm in patterned size-controlled clusters.</a> Stem Cell Res. 6, 276-285 (2011).</li><li>Beattie, G. M., Otonkoski, T., Lopez, A. D., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+and+function+of+human+fetal+pancreatic+cells+after+cryopreservation.">Maturation and function of human fetal pancreatic cells after cryopreservation.</a> Transplantation. 56, 1340-1343 (1993).</li><li>Sandler, S., 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=Tissue+culture+of+human+fetal+pancreas.+Effects+of+nicotinamide+on+insulin+production+and+formation+of+isletlike+cell+clusters.">Tissue culture of human fetal pancreas. Effects of nicotinamide on insulin production and formation of isletlike cell clusters.</a> Diabetes. 38 Suppl 1, 168-171 (1989).</li><li>Otonkoski, T., Beattie, G. M., Cirulli, V., Mally, M. I., Hayek, A., Sharvetnick, N., Landes, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pancreatic regeneration. , (1997).</li><li>Otonkoski, T., Beattie, G. M., Mally, M. I., Ricordi, C., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+is+a+potent+inducer+of+endocrine+differentiation+in+cultured+human+fetal+pancreatic+cells.">Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells.</a> J Clin Invest. 92, 1459-1466 (1993).</li><li>Xie, R., 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=Dynamic+chromatin+remodeling+mediated+by+polycomb+proteins+orchestrates+pancreatic+differentiation+of+human+embryonic+stem+cells.">Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells.</a> Cell Stem Cell. 12, 224-237 (2013).</li><li>Hardikar, A. A., 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=Functional+maturation+of+fetal+porcine+beta-cells+by+glucagon-like+peptide+1+and+cholecystokinin.">Functional maturation of fetal porcine beta-cells by glucagon-like peptide 1 and cholecystokinin.</a> Endocrinology. 143, 3505-3514 (2002).</li><li>Beattie, G. M., Cirulli, V., Lopez, A. D., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+expansion+of+human+pancreatic+endocrine+cells.">Ex vivo expansion of human pancreatic endocrine cells.</a> J Clin Endocrinol Metab. 82, 1852-1856 (1997).</li><li>Beattie, G. 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=Trehalose:+a+cryoprotectant+that+enhances+recovery+and+preserves+function+of+human+pancreatic+islets+after+long-term+storage.">Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.</a> Diabetes. 46, 519-523 (1997).</li><li>Hayek, A., Beattie, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+transplantation+of+human+fetal+and+adult+pancreatic+islets.">Experimental transplantation of human fetal and adult pancreatic islets.</a> J Clin Endocrinol Metab. 82, 2471-2475 (1997).</li><li>Otonkoski, T., Beattie, G. M., Lopez, A. D., Hayek, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+hepatocyte+growth+factor/scatter+factor+to+increase+transplantable+human+fetal+islet+cell+mass.+Transplant.">Use of hepatocyte growth factor/scatter factor to increase transplantable human fetal islet cell mass. Transplant.</a> Proc. 26, 33-34 (1994).</li><li>Beattie, G. 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=A+novel+approach+to+increase+human+islet+cell+mass+while+preserving+beta-cell+function.">A novel approach to increase human islet cell mass while preserving beta-cell function.</a> Diabetes. 51, 3435-3439 (2002).</li></ol>

The authors declare that they have no competing financial interests.

The methods presented here enable one to generate ICCs from human fetal pancreas and subsequently image for markers of cell proliferation and pancreatic endoderm. The process of human fetal pancreas dissociation required about 90 min, followed by a 72 hr ICC formation period, an approach that is substantially different from protocols to isolate &#946; cell progenitor cells from mouse. The protocol presented here provides a reproducible method that allows the researcher to explore human fetal pancreatic development. Two different types of longitudinal studies can be performed. First, the potential to generate functional pancreatic cell types (ductal cells, exocrine cells, and hormone positive cells) from different gestational ages can be assessed after transplantation. Second, ICCs from a single gestational age can be grown in culture, treated with selected pharmacological agents and transplanted at different time points during ICC culture to explore differences in cell fate. With the tremendous efforts currently being directed at hESC differentiation into insulin producing cells, the problems associated with insulin secretion in response to physiological stimuli are again being revived. Human ICCs provide a unique model system to study this critical aspect of fetal pancreas cell proliferation and &#946; cell development.
As with any protocol to isolate cells from tissues, details are critical for success. A number of parameters are unfortunately outside of the control of the laboratory personnel performing the experiment. Two problems encountered by this laboratory include poor quality of starting material and delivery of non-pancreatic tissue. Healthy fetal pancreatic tissue is firm to a scissor cut. If the tissue cuts too easily, it is a sign that the pancreatic enzymes have begun to digest the pancreas itself. From this there is unfortunately no recovery and the preparation is best cancelled. Infrequently, an inexperienced technician removing the pancreas will confuse sections of the gut for the pancreas. These samples will have much more elasticity than a pancreas and a notable lumen will be present. Again, these samples should be discarded.
When the protocol above is followed as described, there are rarely any issues that arise to throw the isolation off track. A few points to remember to ensure a smooth isolation include, to use sharp scissors for precise pancreas cutting and to make sure to not leave any tissue sample too big to digest. If the cuts are not small enough, then the collagenase does not work effectively, and few ICCs are formed. Conversely, when using a new batch of collagenase, start with a similar level if IU (international units) for digestion as previously used. However, decrease the incubation time to ensure that over digestion does not occur. This troubleshooting technique ensures that the IU label does not vary significantly from batch to batch.
Taken in perspective, this technique for isolation of human fetal ICCs is relatively standard in the field. Most laboratories confirmed our findings that addition of HGF to the media helps the cells survive and proliferate33. In summary, we have developed a method to isolate human fetal ICCs fresh pancreata with gestational ages ranging from 9 to 23 weeks. The cells can be grown as a monolayer or in suspension for experiments to visualize pancreatic endocrine transcription factors and human insulin.

A primary limitation to cell-based insulin replacement therapies in type 1 diabetes is the paucity of human islets available for transplantation. The ability to regulate proliferation and differentiation of human pancreatic precursor cells into insulin-producing cells that meet the metabolic demands of an insulin-deficient state remains a critical building block for a cell-based therapy to treat type 1 diabetes.
Until the advent of hESC derived insulin-producing cells, human fetal pancreatic endocrine cells or their precursors were viewed as potential sources of cells for clinical transplantation. Although the scientific and regulatory landscape has changed in the past few years, there remains a vital need to understand how the human fetal pancreas develops. Many now view therapeutic use of human fetal cells as unlikely, however if effective and safe methods to expand the cells were established, therapeutic use of these cells could again be explored. A major hurdle that remains is that in vitro transformation of human fetal pancreatic cells aggregates (ICCs) into glucose responsive, insulin secreting endocrine cells is currently an inefficient process. Although much work over almost 30 years has elucidated and delineated the expression profile of transcription factors required for the development of endocrine pancreas, there remain gaps in our knowledge about how temporal expression of transcription factors is regulated and related to cell function.
Until the advent of hESC derived insulin-producing cells, human fetal pancreatic endocrine cells or their precursors were viewed as potential sources of cells for clinical transplantation. Although the scientific and regulatory landscape has changed in the past few years, there remains a vital need to understand how the human fetal pancreas develops. Many now view therapeutic use of human fetal cells as unlikely, however if effective and safe methods to expand the cells were established, therapeutic use of these cells could again be explored. A major hurdle that remains is that in vitro transformation of human fetal pancreatic cells aggregates (ICCs) into glucose responsive, insulin secreting endocrine cells is currently an inefficient process. Although much work over almost 30 years has elucidated and delineated the expression profile of transcription factors required for the development of endocrine pancreas, there remain gaps in our knowledge about how temporal expression of transcription factors is regulated and related to cell function.
Recently, the stem cell field has employed the accumulated knowledge about temporal transcription factor expression during islet development to drive the production of cells that express the markers of mature endocrine cells. Although genesis of insulin producing cells from hESC and induced pluripotent cells (iPSC) has made substantial and significant advances in the past few years, the most effective protocols require two distinct differentiation phases: 1) an early in vitro differentiation to generate cells expressing pancreatic precursor transcription factors, followed by 2) in vivo maturation after transplant &#8212; a so called &#8220;black box&#8221; period. To move forward, advances in understanding the biology underlying islet maturation in vivo, regardless of cell source, must be understood at a biochemical level. The similarity between the results obtained with ICCs and hESC suggests that a number of critical biochemical processes that regulate the transition of human pancreatic precursor cells into mature, glucose responsive, insulin secreting cells in vitro remain unknown. A central part of this understanding will be to develop novel methodologies to derive functional endocrine cell populations from pancreatic progenitor cells and hESC will require not only biochemical approaches that elucidate the maturation events, but methods to analyze the changes.
Why do we believe that imaging human fetal pancreatic cells is a critical aspect of identifying changes in islet maturation? The answer lies partially in the historical quest to generate insulin producing cells. Both model and tissue-culture systems for pancreatic precursors and islets have allowed researchers to explore the significant differences that exist between maturation of animal islets and human islets&#160;in vitro. A limitation to the exploration of human fetal pancreas development is that the gestational ages that can be legally used only give rise to heterogeneous cell aggregates. Although the isolated fetal human cells resemble islets, staining after in vitro culture from gestational ages 9-23 weeks reveals less than 15% contain markers for endocrine cells, with most cells not staining for islet hormones. However, after transplantation and in vivo maturation, a large majority of cells express endocrine markers 6,25. The results from these studies are being echoed today with hESC differentiation protocols that are only able to generate modest populations of single hormone positive endocrine cells after in vitro differentiation. In vitro methods to enhance populations of hormone positive cells will likely provide insight into in vivo islet maturation. Furthermore, understanding the molecular events that drive human fetal pancreatic development will likely improve efforts to derive insulin producing cells from hESC and further delineate the mechanisms that regulate &#946;&#160;cell regeneration and pancreatic cell transdifferentiation.
The similarities between human fetal pancreatic cell and hESC maturation can be extended to cell function. Like murine cells, both human fetal cells and differentiated hESC are unable to release insulin in response to glucose after islet neoformation&#160;in vitro26,27. However, upon transplantation into nude mice and maturation, both human islet-like clusters and insulin producing cells derived from hESC exhibit an endocrine phenotype. Three months after grafting, almost 90% of the transplanted cells from either population are insulin positive, and function normally as determined by the release of C-peptide17,26. These results indicate that transplantation provides context and unidentified cues that promote islet maturation. Optimized imaging protocols, such as the one described here, for human fetal pancreatic cells will aid in the search to identify factors that modulate and accelerate maturation. Fundamental biochemical exploration of human fetal pancreatic cells and hESC differentiated towards an endocrine lineage at this stage of development is essential for measuring efficiency of maturation.
The following protocol, outlined in Figure 1, provides our current method for the isolation of human fetal pancreatic cells, called ICCs from whole human fetal pancreas and imaging of these cells. This protocol requires an initial preparation of cells from tissue, which can be subsequently grown as a monolayer or in suspension. Preparation of cells for imaging of commonly used markers of endocrine cell proliferation and maturation is described.
Generation and imaging of ICCs in the absence or presence of a variety of chemical modifying agents provides a rapid method, compared with transplantation models, to help identify culture conditions and compounds that accelerate maturation of human fetal pancreatic cells into fully functional exocrine, ductal, or hormone producing pancreatic cells.

<table border="0" cellpadding="0" cellspacing="0" width="1000">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Trehalose SG</td>
			<td style="width:219px;">Hayashibara</td>
			<td style="width:115px;">Trehalose SG</td>
			<td style="width:319px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase XI&#160;</td>
			<td>Sigma</td>
			<td>C-9407</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Life Technologies</td>
			<td>24020-117</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 with glutamate</td>
			<td>Life Technologies</td>
			<td>11879020</td>
			<td>no glucose or HEPES</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human AB Sera, Male Donors</td>
			<td>Omega Scientific</td>
			<td>HS-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fungizone</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentamicin Solution 50 mg/ml</td>
			<td>Invitrogen</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1M Hepes, pH 7.0</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin - Streptomycin 100X Solution</td>
			<td>Life Technologies</td>
			<td>15070063</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamax</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hepatocyte Growth Factor (HGF)</td>
			<td>Peprotech</td>
			<td>100-39</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GLP-1</td>
			<td>Peprotech</td>
			<td>130-08</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Invitrogen</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dnase</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Petri Dishes, 60 x 15 mm; Stackable, venting ribs</td>
			<td>Spectrum Laboratory Products, Inc.</td>
			<td>D210-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey Serum&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BrdU</td>
			<td>Life Technologies</td>
			<td>00-0103</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-insulin (mouse monclonal; 1:1000)</td>
			<td>Sigma</td>
			<td>I2018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-glucagon (mouse monoclonal; 1:2000)</td>
			<td>Sigma</td>
			<td>G2654</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 (mouse monoclonal)</td>
			<td>Novus Biologicals</td>
			<td>NBP1-47910</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 (goat polyclonal)</td>
			<td>AbCam</td>
			<td>47383</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDX1 (rabbit polyclonal)</td>
			<td>AbCam</td>
			<td>47267</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 546 (rabbit)</td>
			<td>Invitrogen</td>
			<td>A11010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 546 (mouse)</td>
			<td>Invitrogen</td>
			<td>A11003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 (rabbit)</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AlexaFluor 488 (mouse)</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ki67</td>
			<td>Lab Vision Neomarkers</td>
			<td>RM 9016-50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pan-CK (mouse monoclonal; 1:100)</td>
			<td>Immunotech</td>
			<td>2128</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CK19</td>
			<td>AbD Serotech</td>
			<td>MCA 2145</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Cell Signaling</td>
			<td>4083</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HTB9 cell line</td>
			<td>ATCC</td>
			<td>5637</td>
			<td>see Beattie 1997 reference to generate matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agarose</td>
			<td>Sigma</td>
			<td>A-6013</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, culture, and imaging of human fetal pancreatic cell clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

Careful preparation of fetal ICCs is critical to the success of this protocol. Representative pictures of how the cells typically look at defined periods after plating are shown in Figure 2. After purification, the freshly plated cell aggregates appear as small, sparse clear clusters that contain few cells (Figure 2A). After the first 24 hr (Figure 2B), many of the cell clusters become larger, but numerous small clusters remain. By 48 hr, the clusters have expanded significantly, but remain asymmetrical (Figure 2C). At 72 hr, the ICCs should be clear, relatively uniform in size and shape (Figure 2D). It is at this point that the cells can be either plated on matrices, such as HTB-9, for 24 hr prior to immunofluorescence or allowed to grow for another 72 hr in the absence or presence of chemicals or drugs that may alter expression of genes required for pancreatic endocrine cell generation. Figure 3 highlights a number of commonly used antibodies to assess either ICC proliferation or differentiation towards pancreatic endoderm. In Figure 3A, ICCs were plated on HTB-9, grown for 4 days, and stained for PDX1, a critical marker of pancreatic development. Although in vitro staining of ICCs is performed routinely in our laboratory, more often, human fetal ICCs are transplanted under the kidney capsule of nude mice and allowed to proliferate and differentiate in vivo6,9,30-32. At specified times after transplantation, mice are sacrificed and the kidney containing the transplanted ICCs is removed, fixed in 4% paraformaldehyde, and embedded in paraffin. Sequential 5-&#956;m sections are generated either on site using a microtome or by a core microscopy facility. Epitope unmasking and staining of proteins from paraffin embedded tissue for immunofluorescent microscopy is described in optional protocol 3.10-3.17. In Figure 3B, transplanted ICCs were stained with the epithelial cell marker pan cytokeratin (PanCK; green) and with Ki67 (red) to assess cell proliferation. In another example, both human insulin (green) and glucagon (red) were detected after transplantation and maturation under the kidney capsule of a nude mouse (Figure 3C).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50796/50796fig1highres.jpg" width="500" /> 
Figure 1. Flow chart of human fetal ICC preparation and staining for Immunofluorescence.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50796/50796fig2highres.jpg" width="500" /> 
Figure 2. Representative fetal ICCs preparation at 0, 24, 48, or 72 hr after plating. (A) ICCs in suspension were imaged immediately after plating, (B) 24 hr after plating, (C) 48 hr after plating, or (D) 72 hr after plating. Scale bar for A-C, 10X magnification = 300 &#181;m, scale bar for D, 20 magnification = 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/50796/50796fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/50796/50796fig3highres.jpg" /> 
Figure 3. Immunofluorescence of plated ICCs. Routinely, ICCs are imaged for (A) the pancreatic endoderm marker PDX1 (green), (B) endocrine cells (pan-CK; green) and proliferation (Ki67; red), (C) insulin (green) and glucagon (red). Scale bar for A-C, 40X magnification = 20 &#956;m.

Watch this Scientific Journal Video about Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters at JoVE.com

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

isolation-culture-and-imaging-of-human-fetal-pancreatic-cell-clusters

Research

JoVE Journal

Medicine

14.0K Views.  University of California, San Diego.  Un protocollo per isolare, i cluster cultura e l'immagine delle cellule insulari (CICS) derivate da cellule pancreatiche fetali umane è descritto. Il metodo in dettaglio i passi necessari per generare ICCs dal tessuto, la cultura come monostrati o in sospensione come aggregati, e immagine per i marcatori di proliferazione e decisioni destino delle cellule pancreatiche. 

Video: Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...