The authors would like to thank Dr. Klaus Kaestner for the gift of murine Ngn3-EGFP embryonic stem cell line, Mrs. Lucy Brown and the Analytical Cytometry Core for assistance in cell sorting. This work was funded in part by NIH grants R21DK069997 and R01DK081587 awarded to H.T.K. and from California Institute for Regenerative Medicine (CIRM) training grants awarded to M.W. and L.S.

1. Differentiation of murine ES cells to pancreatic-like cells in vitro to generate conditioned media (CM) for colony assay
Procedures for murine ES cell maintenance, embryoid body (EB) formation in suspension culture, and attachment culture for further differentiation have been previously described1-3 and are not the focus of this report. A schematic presentation of the steps involved in our differentiation protocol is shown in Figure 1.
In order to obtain high-quality conditioned media, it is important to first generate 6-day-old EBs in which at least 30-40% of them are with diameters of at least 150 &mu;m. The larger EBs should contain dark spots under the light microscope, suggesting enhanced differentiation (Figure 2). Selection of high quality fetal calf serum (FCS) is critical to generate such EBs. High quality FCS lots are selected based on their ability to support the differentiation of EBs towards insulin 1, glucagon and amylase 2A gene expression in the later stages (day 18-20) as detected by RT-PCR analysis. Furthermore, when day-6 EBs are transferred from suspension culture to attachment culture (approximately 80 - 100 EBs per well in 6 well plates) it is important to gently rotate the plates to gather the day-6 EBs into the center of the wells. For reasons unknown, maneuvers that enhance cell-cell contacts result in better development of pancreatic-like cells from EBs in the subsequent attachment culture stage.
<ol>
 <li>On culture day 13, replace the attachment culture media (DMEM/F-12 (1:1), 15% knockout serum replacement, 2 mM L-glutamine, 50 U/mL penicillin, 50 &mu;g/mL streptomycin) with fresh attachment culture media containing10 mM nicotinamide, 0.1 nM exendin-4, and 10 ng/mL human recombinant activin &szlig;B (all concentrations are final).</li>
 <li>On culture day 16, collect the media from step 1.1 and filter it through a 0.2 &mu;m polyethersulfone membrane. From this point forward the collected media will be referred to as conditioned media (CM). Aliquot the CM into 15 mL Falcon tubes and store it at -80&deg;C until just prior to semi-solid culture (see Section 3 below).</li>
</ol>
2. Obtaining pancreatic-like progenitors in single cell suspension using a fluorescence activated cell sorter
A knock-in murine ES cell line,4 designated as Ngn3-EGFP, where the EGFP reporter gene replaced one allele of the Ngn3 locus was differentiated as previously described.2 Ngn3 is a basic helix-loop-helix (bHLH) containing transcription factor expressed by pancreatic endocrine progenitors during development.5 Day-16 cultures were normally sorted to obtain differentiated Ngn3-EGFP+ and Ngn3-EGFP- cells.1,2
<ol>
 <li>Dissociation of differentiated cells into a single cell suspension.
 <ol>
 <li>Incubate day-16 cultures with 0.25% trypsin-EDTA (3 min, 37&deg;C) to dislodge the cells from culture wells.</li>
 <li>Add 10% FCS to stop trypsin activity.</li>
 <li>Wash cells with magnesium and calcium-free phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA), and centrifuge at 300 x g for 5 min.</li>
 <li>Remove supernatant and incubate the cell clumps with 4 mg/mL collagenase B and 2000 U/mL deoxyribonuclease (DNase) I (30 min, 37&deg;C) to produce a single cell suspension. Use a 1000 &mu;L pipette to disrupt the cell pellets every 10 min to hasten the dissociation process.</li>
 <li>Wash cells with PBS containing 0.1% BSA, and centrifuge at 300 x g for 5 min.</li>
 <li>Remove supernatant and resuspend the cells in PBS containing 0.1% BSA.</li>
 </ol>
 </li>
 <li>Preparation of cells for sorting.
 <ol>
 <li>Add 2 &mu;L DNase I (1 MU/ mL) for every mL cell suspension to prevent cell reaggregation during sorting.</li>
 <li>Pass the single cell suspension through a 40 to 70 &mu;m mesh to remove large cell aggregates.</li>
 <li>Add DAPI (1 &mu;g/mL final concentration) to the single cell suspension.</li>
 <li>Acquire flow cytometry data on a cell sorter. A MoFlo MLS (Beckman Coulter) cell sorter was used for this report.</li>
 <li>Gating of cell populations for sorting. Ngn3-EGFP+ and Ngn3-EGFP- cells are gated first with forward scatter vs. side scatter (Figure 3A), followed by DAPI vs. side scatter (Figure 3B), pulse width vs. side scatter (Figure 3C), and finally FL1 vs. auto-fluorescence (Figure 3D). The final sort gate of Ngn3-EGFP+ cells is intentionally moved to the right (Figure 3E; arrow) in order to limit contamination of Ngn3-EGFP- cells during sorting. For negative control gating, either day-6 EBs from Ngn3-EGFP ES line or the same stage, day-16 culture from a non-transgenic line, such as TL1 ES cells (Figure 3D, left panel), can be used.</li>
 </ol>
 </li>
 <li>Sort Ngn3-EGFP+ and Ngn3-EGFP- cells.</li>
</ol>
3. Plating sorted cells to semi-solid culture
<ol>
 <li>Matrigel preparation.
 <ol>
 <li>Aliquot the Matrigel to 1 mL per tube and store at -20&deg;C before use.</li>
 <li>Frozen aliquots should be placed at 4&deg;C overnight or on ice for 3 hours to thaw just prior to use. Thawed aliquots must remain on ice during semi-solid culture preparation to avoid resolidification.</li>
 </ol>
 </li>
 <li>Methylcellulose solution (3.3%) preparation. 
 Methylcellulose powder cannot be autoclaved. In order to obtain bacteria-free methylcellulose solution, use clean weighing boats and autoclaved stir bars, beakers, flasks and other hardware during preparation process.
 <ol>
 <li>One day before the methylcellulose solution preparation, store 200 mL of sterile double distilled water at 4&deg;C. Also, prepare 500 mL of 2x DMEM/F12 media (prepare by dissolving powdered DMEM/ F-12 in doubled distilled water) containing 100 U/mL penicillin and 100 &mu;g/mL streptomycin and store at 4&deg;C.</li>
 <li>On the day of the methylcellulose preparation, add 500 mL of sterile double distilled water to a 1000 mL glass beaker with a piece of aluminum foil on top. Boil for at least 30 min to rid the water of air bubbles.</li>
 <li>Place a large sized stir bar (at least 3 inches in length) into a 2000 mL glass flask.</li>
 <li>Measure 300 mL of boiling water and transfer it to the flask.</li>
 <li>Place the flask on a stir plate (without heating). Slowly stir the hot water to avoid generating air bubbles.</li>
 <li>Weigh 33 grams of methylcellulose powder and very slowly add it to the hot water. Hot water is necessary to wet the methylcellulose powder efficiently and aid the subsequent dissolution in cold temperature.</li>
 <li>Continue to stir the powder until the temperature is reduced to approximately 40-45&deg;C.</li>
 <li>Add the 200 mL cold sterile double distilled water to the cooling methylcellulose mixture. Immediately after, you should start to see the solution turn transparent and viscous, indicating that methylcellulose is dissolving into the aqueous phase. Hand swirl the flask to ensure the solution is mixed thoroughly.</li>
 <li>Add the 500 mL cold 2x DMEM/F12 media. Continue to swirl by hand to mix.</li>
 <li>Place the flask on a stir plate in a cold room, and slowly stir the methylcellulose solution for 24-48 hours.</li>
 <li>Aliquot the solution into 125 mL per storage bottle, and store at -20&deg;C. A sample should be dispensed into a sterile petridish and placed in 37&deg;C incubator to test sterility. Thawed methylcellulose solution can be kept in refrigerator for up to 2 months.</li>
 </ol>
 </li>
 <li>Conditioned media preparation. 
 Thaw CM (see section 1) just prior to use. Keep the CM on ice throughout semi-solid culture preparation.</li>
 <li>Prepare the following growth factors: 1.0 M nicotinamide, 0.1 &mu;M exendin-4, 10 &mu;g/mL recombinant human activin &beta;B, and 10 &mu;g/mL VEGF-A. Store the nicotinamide and exendin-4 at -20&deg;C and activin &beta;B and VEGF-A at -80&deg;C, and thaw them immediately before use. All semi-solid culture media components should be kept on ice during plating. It should be noted that addition of VEGF-A was later found not necessary for colony formation.2</li>
 <li>Fetal calf serum preparation. FCS should be heat inactivated before use. Incubate the FCS for 30 minutes at 56&deg;C in a water bath to inactivate complement proteins. If the initial FCS stock bottle holds more than 500 mL aliquot it into 125 mL vials. The increased surface area will ensure even and thorough heating of the entire volume. Allow the bottles to cool at room temperature for at least 1 hour before storing at -20&deg;C. Thawed FCS is 0.2&mu;m filtered before use.</li>
 <li>Mix culture components with sorted cells. 
 Keep the cells and all semi-solid media components on ice during preparation at all times to prevent solidification of Matrigel. All supplies that come in contact with Matrigel including, pipette tips, syringes and needles, should be cold. Place these supplies at -20&deg;C at least half an hour before use.
 <ol>
 <li>Determine the quantity of cells to be seeded per well in a 24-well plate. Up to 25,000 cells can be plated per 24-well. One could perform a titration of cell density in initial experiments to determine the optimal seeding cell number.</li>
 <li>Add the culture components to a 5 mL polystyrene tube with a snap cap in the sequence described in Table 1 (note that the cells are added last to avoid premature stimulation). Add the 3.3 % methylcellulose to the polystyrene tubes first and the sorted cells last. Note that the 3.3 % methylcellulose solution needs to be drawn up and added to the polystyrene tubes with a 16 &frac12; gauge needle and an adequately marked graduated syringe. In general, in order to correctly measure the volume of viscous solutions, such as the 3.3 % methylcellulose solution and the final culture mixture, it is important to aspirate some solution in the syringe first, and then immediately push the solution out to expel the air. The 3.3 % methylcellulose solution may be warmed to room temperature if it is too difficult to draw out. However, ensure it is cooled on ice after it is dispensed into the polystyrene tubes and before the addition of Matrigel.</li>
 </ol>
 </li>
 <li>Plate the semi-solid media containing sorted cells.
 <ol>
 <li>After ensuring the caps of the polystyrene tubes are firmly in place, shake the tubes by hands vigorously and thoroughly. Air bubbles will be generated during this process.</li>
 <li>Place the tubes on ice for 5 min or until the small air bubbles surface.</li>
 <li>Use a 1mL syringe with an 16 &frac12; or 18 &frac12; -gauge needle to aspirate the contents. Follow the advice in 3.6.2 to draw accurate volume of viscous solution without air in syringes.</li>
 <li>Slowly dispense 500 uL of media into each 24-well. Use the low attachment 24-well plates only. Add the media directly to the center of the well. The semi-solid media will automatically spread out in the wells. For each experimental sample, quadruplicated wells are routinely used in order to obtain statistically significant results. Only the 4 innermost columns of a horizontally oriented 24-well plate are used for culture. Fill the remaining wells with sterile water to aid humidification of the cell-containing culture.</li>
 <li>Incubate the cells in 37&deg;C and 5% CO2 air.</li>
 <li>Delayed addition of growth factors. It is possible to add growth factors after the initiation of the culture. Such maneuvers may be used to address the survival effects of certain growth factors. Growth factor solutions up to 100 &mu;L per well may be dripped via a 1 mL syringe with a 26 G 3/8 needle and allowed to diffuse into the semi-solid media.</li>
 </ol>
 </li>
</ol>
4. Observing colony formation under the light microscope
Cells should be observed immediately after plating to ensure that single cells are randomly and evenly distributed throughout out the wells. If most of the cells or the resulting colonies are distributed at the margin of the wells, this will indicate that dispensing the semi-solid media into the wells was too forceful (see section 3.7.4). Due to the meniscus effect colony density at the margins of the culture wells may be higher compared to those residing in the center.
<ol>
 <li>Colony number quantification. Due to the 3-dimensional feature of the media, the colonies will appear at different focal points. Thus, an adjustment of the focus for each colony may be required during observation by light microscopy. Attach a grid under the 24-well when counting in order to avoid registering the same colony twice. A grid may be obtained by cutting the wall of a NUNC petridish (Cat. No.169558). A 10x objective lens on a light microscope should be used to observe and count the colonies.</li>
</ol>
5. Representative Results:
Previous experiments had shown that starting with 2.5 x 105 murine R1 ES cells (in 50 mL media total) one could expect to generate approximately 5000 appropriate sized day-6 EBs. The Ngn3-EGFP ES cells were less efficient; 4 times more cells were required to generate similar-sized EBs by day 6. Because 25 day-6 EBs contain an average of 1.88 &plusmn; 0.67 x 105 cells,1 approximately 37.6 x 106 cells can be obtained from 5000 EBs. During attachment culture the cells are expected to expand 2.72 &plusmn; 0.32 fold.1 Thus, 5000 day-6 EBs will yield approximately 100 x 106 day-16 cells before sorting. After gating, the Ngn3-EGFP+ cells will make up approximately 10 % of the total day-16 cell population (Protocol Text Section 2.2.5 and Figure 3).
Example of the photomicrographs of insulin-expressing colonies derived from murine ES cells is shown in Figure 4. These colonies have a distinctive morphology; small (~60-100 &mu;m) round colonies with individual cells in the colonies that are small, dark, and not light reflective. In our previous publication,2 real-time RT-PCR and double immunofluorescent staining of individually hand-picked colonies revealed expression of insulin, C-peptide and glucagon. These results demonstrate the ability of single Ngn3-EGFP+ cells to differentiate into cells containing at least two islet hormones simultaneously, resembling primitive first-wave like endocrine cells.
The colony assay for dispersed single cells enables the study for those progenitors that have the capacity to initiate proliferation and differentiation in vitro (Figure 5). Thus, this is an assay that can measure the intrinsic function of individual progenitor cells. The progenitors that have the capability of becoming insulin-expressing colonies are termed insulin-expressing colony forming units (ICFUs) (Figure 5). Consistent to the idea that Ngn3 marks endocrine progenitor cells in the developing pancreas,5 Ngn3-EGFP+, but not Ngn3-EGFP- cells from mES cell-derived populations are enriched for the ICFUs.2 However, the frequency of these progenitors is still low; approximately 0.1 % to 0.6 % of the total Ngn3-EGFP+ population isolated from day 16 culture are ICFUs.2 Regardless, a clear difference in colony-forming efficiency is expected, with Ngn3-EGFP+ cells the highest, followed by the presorted cells, and then the Ngn3-EGFP- cells.
<img alt="Figure 1" src="/files/ftp_upload/3148/3148fig1.jpg" /> 
Figure 1. Timeline of in vitro murine ES cells differentiation to pancreatic-like cells. For conditioned media (CM) generation, murine ES cells are grown in suspension with 15 % FCS for the first six days with decreasing doses of monothioglycerol (6.0 x 10-3 M for the first two days and 6.0 x 10-4 M for the following four days) to generate EBs. Day-6 EBs (80-100 per well) are then transferred to attachment 6-well dishes containing 15 % knockout serum replacement. 5% FCS may be added between days 6-10 of culture to hasten the attachment of EBs. Nicotinamide, exendin-4, and human recombinant activin &beta;B are then added to the media on culture day 13 (see section 1.1). Media is collected on culture day 16 and at this point has become conditioned media. Day 16 cells are then sorted and plated into semi-solid media for colony assay.
<img alt="Figure 2" src="/files/ftp_upload/3148/3148fig2.jpg" /> 
Figure 2. Representative photomicrograph of large day-6 embryoid bodies derived from murine embryonic stem cells. Ideal day-6 EBs are at least 150 &mu;m in diameter and have darkened centers (arrows). Expression of early pancreatic progenitor markers (such as Pdx-1 and Sox9) is enhanced in these EBs (data not shown).3
<img alt="Figure 3" src="/files/ftp_upload/3148/3148fig3.jpg" /> 
Figure 3. Sorting gates for mES cell-derived day-16 Ngn3-EGFP expressing cells. (A) Forward and side scatter gates, indicative of cell size and granularity respectively, eliminate non-cellular debris. (B) DAPI is used to identify dead cells, which will be stained positive when excited at 405-413 nm and emission detected with a 450/40 filter. (C) Pulse width, the width of the electronic forward scatter pulse, reveals doublets, which should be eliminated before being passed through the fluorescence activated cell sorter. (D) Gating of EGFP (channel FL1) versus autofluorescence using day-16 cells from a control parental cell line such as TL1 (left panel) illuminates true fluorescence and thus the Ngn3-EGFP+ cell population (right panel). (E) Red arrow shows manual right-shift of EGFP+ gate just before sorting to increase separation of false positive cells. Events in both (D) and (E) are gated based on regions (R1 - R3) shown in (A-C).
<img alt="Figure 4" src="/files/ftp_upload/3148/3148fig4.jpg" /> 
Figure 4. Representative photomicrographs of insulin-expressing colonies in semi-solid culture. Colonies are randomly distributed and appear as small dark, non-light reflective clusters. Individual colonies can later be assayed for gene and protein expression by RT-PCR and immunohistochemistry analyses, respectively (not shown; see video).
<img alt="Figure 5" src="/files/ftp_upload/3148/3148fig5.jpg" /> 
Figure 5. A colony assay that allows functional and quantitative assessments of single progenitor cells. Numeration of the resulting colonies is used to calculate the frequency of the progenitor cells among the total plated cells. The composition of cells within each colony is indicative of the lineage potential of the initiating progenitor. Using this assay, we found that Ngn3-EGFP+ cells derived from murine embryonic stem cells were enriched for insulin-expressing colony-forming units (ICFUs), progenitor cells that have the capability to differentiate into insulin-expressing colonies.2
<img alt="Table 1" src="/files/ftp_upload/3148/3148table1.jpg" /> 
Table 1. Semi-solid Culture Media Components and Order of Addition.

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We have nothing to disclose.

Colony assays that allow quantitative and functional assessments of individual pancreatic progenitors have been limited so far, which greatly hampered the progress in the studies of pancreatic stem and progenitor cell biology. The essence of a colony assay is that it measures the intrinsic ability of progenitor cells, rather than solely their phenotype, allowing the functionality and lineage potentials of the progenitor cells to be observed. It is also a quantitative tool that can determine the number of progenitors in a given population. In the hematopoietic stem cell field, colony assays have played important roles in identification, as well as deciphering the growth factor requirements, interactions, and mechanisms governing the lineage commitment of these cells.6 In the field of pancreatic progenitor cell biology, pancreatic organ7,8 or bulk cultures9-13 have been devised, however, such assays do not address biological questions at the single cell level. Without single cell analyses, the existence of multi-potential pancreatic stem/progenitors cannot be proven unequivocally.14
Here we show that insulin-expressing colony-forming progenitors can be assayed using a three-dimensional, Matrigel-containing, semi-solid culture system. Although a suspension culture system15,16 has been developed which permits single adult pancreatic progenitors to proliferate and differentiate into "pancreatospheres", our colony assay provides additional advantages. First, our semi-solid culture media system allows plating of up to 25,000 sorted cells per 500 &mu;L culture media per 24-well while maintaining the resulting colony formation in the linear range.2 In contrast, the suspension pancreatosphere assay requires the use of single cell deposition method from the cell sorter to allow single cell analyses, and thus a larger volume of culture media and incubator space are required. Therefore, our colony assay is relatively easy and cost-effective to conduct when large numbers of different cells need to be compared simultaneously for progenitor activities. Second, our semi-solid media allows uniform distribution of extracellular matrix components and growth factors, which allows both proliferation and differentiation to occur in the same well. In contrast, it is more difficult to provide such an environment, especially the matrix, to cells in suspension culture. We are aware that with our method it cannot be definitively proved that each colony is derived from a single cell, as two progenitors may be randomly positioned near each other and form one colony. However, this problem can be overcome by secondary analysis using single cell manipulation. Direct hand-picking of sorted single cells could definitively demonstrate the single cell origin of each colony.
In summary, the insulin-expressing colony-forming activity of progenitors in semi-solid media described herein provides unique opportunities for mechanistic studies of pancreatic progenitors at the single cell level. For example, in a prior report we found that addition of Matrigel, a crude mixture of extracellular matrix proteins, into the culture media is required for the formation of the insulin-expressing colonies from Ngn3-EGFP+ cells,2 suggesting the extracellular matrix is important for those progenitors. Using this colony assay, it will now be possible to further dissect and define specific matrix requirements for such activity. In addition to studying progenitors derived from mouse embryonic stem cells, we are currently investigating whether this in vitro assay system also supports formation of other distinctive colonies derived from the pancreas and liver of perinatal and adult mice, as well as from human cadaveric pancreatic tissue devoid of islets.

<table border="1">
<tbody>
	<tr>
		<td>Material/ Reagent</td>
		<td>Company</td>
		<td>Catalogue Number</td>
		<td>Comments</td>
	</tr>
	<tr>
		<td>1X DMEM/ F-12 (1:1)</td>
		<td>Mediatech</td>
		<td>15-090-CV</td>
		<td></td>
	</tr>
		<tr>
		<td>Fetal Calf Serum</td>
		<td>Tissue Culture Biologicals</td>
		<td>201</td>
		<td>Refer to Section 1.1</td>
	</tr>
		<tr>
		<td>L-glutamine</td>
		<td>Gibco</td>
		<td>25030-081</td>
		<td></td>
	</tr>
		<tr>
		<td>1X Dulbecco's PBS</td>
		<td>Mediatech</td>
		<td>21-031-CV</td>
		<td></td>
	</tr>
		<tr>
		<td>Bovine Serum Albumin</td>
		<td>Sigma</td>
		<td>A8412</td>
		<td>7.5 &#37;</td>
	</tr>
		<tr>
		<td>Matrigel</td>
		<td>Becton Dickinson</td>
		<td>356231</td>
		<td>Reduced Growth Factor</td>
	</tr>
		<tr>
		<td>Methylcellulose</td>
		<td>Sinetsu Chemical, Tokyo, Japan</td>
		<td></td>
		<td>1,500 centipoise (high-viscosity)</td>
	</tr>
		<tr>
		<td>Nicotinamide</td>
		<td>Sigma</td>
		<td>N0636</td>
		<td></td>
	</tr>
		<tr>
		<td>Human Recombinant Activin &beta;B</td>
		<td>R &amp; D Systems</td>
		<td>659-AB-025</td>
		<td></td>
	</tr>
		<tr>
		<td>Exendin-4</td>
		<td>Sigma</td>
		<td>E7144</td>
		<td></td>
	</tr>
		<tr>
		<td>Human Recombinant VEGF-A</td>
		<td>R &amp; D Systems</td>
		<td>293-VE-010</td>
		<td></td>
	</tr>
		<tr>
		<td>24-well plates</td>
		<td>Costar</td>
		<td>3473</td>
		<td>Ultra-low Attachment</td>
	</tr>
		<tr>
		<td>60mm Petri Dish with Grid</td>
		<td>Nunc</td>
		<td>169558</td>
		<td></td>
	</tr>
</tbody>
</table>

a quantitative assay for insulin-expressing colony-forming progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

Watch this Scientific Journal Video about A Quantitative Assay for Insulin-expressing Colony-forming Progenitors at JoVE.com

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

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JoVE Journal

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13.4K Views.  California State University Channel Islands. A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

Video: A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Identification and Analysis of Mouse Erythroid Progenitors using the CD71/TER119 Flow-cytometric Assay

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of red cell formation is regulated by the hormone erythropoietin (Epo), whose synthesis is triggered by tissue hypoxia. A threat to adequate tissue oxygenation results in a rapid increase in Epo, driving an increase in erythropoietic rate, a process known as the erythropoietic stress response. The resulting increase in the number of circulating red cells improves tissue oxygen delivery. An efficient erythropoietic stress response is therefore critical to the survival and recovery from physiological and pathological conditions such as high altitude, anemia, hemorrhage, chemotherapy or stem cell transplantation. 
The mouse is a key model for the study of erythropoiesis and its stress response. Mouse definitive (adult-type) erythropoiesis takes place in the fetal liver between embryonic days 12.5 and 15.5, in the neonatal spleen, and in adult spleen and bone marrow. Classical methods of identifying erythroid progenitors in tissue rely on the ability of these cells to give rise to red cell colonies when plated in Epo-containing semi-solid media. Their erythroid precursor progeny are identified based on morphological criteria. Neither of these classical methods allow access to large numbers of differentiation-stage-specific erythroid cells for molecular study. Here we present a flow-cytometric method of identifying and studying differentiation-stage-specific erythroid progenitors and precursors, directly in the context of freshly isolated mouse tissue. The assay relies on the cell-surface markers CD71, Ter119, and on the flow-cytometric 'forward-scatter' parameter, which is a function of cell size. The CD71/Ter119 assay can be used to study erythroid progenitors during their response to erythropoietic stress in vivo, for example, in anemic mice or mice housed in low oxygen conditions. It may also be used to study erythroid progenitors directly in the tissues of genetically modified adult mice or embryos, in order to assess the specific role of the modified molecular pathway in erythropoiesis.

A flow-cytometric method for identification and molecular analysis of differentiation-stage-specific murine erythroid progenitors and precursors, directly in freshly &#x2013;harvested mouse bone marrow, spleen or fetal liver. The assay relies on cell-surface markers CD71, Ter119, and cell size.

The study of erythropoiesis aims to understand how red cells are formed from earlier hematopoietic and erythroid progenitors. Specifically, the rate of ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...