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This video demonstrates a procedure for generating three dimensional cultures that allow pancreatic like progenitors to differentiate into insulin expressing colonies. First, differentiated cells derived from a neurogen in three EGFP Knockin murine embryonic stem cell line are dissociated and sorted for EGFP expression to enrich for endocrine like progenitors. The EGFP positive sorted single cells are plated into a semi-solid media containing matrigel and growth factors.

This limits the movement of individual progenitor cells while permitting their survival, proliferation and differentiation in vitro. Next, the number resulting colonies with distinctive morphology accounted and scored under a microscope in order to calculate the frequency of colony forming progenitors in a given sorted population. Analysis of individual colonies by real-time PCR and immunofluorescence microscopy reveals insulin expression in colonies derived from ngn three positive cells, but not from NGN three negative cells.

The main advantage of using this technique over existing techniques such as pancreat spheres in suspension culture is that this technique allows for a larger amount of cells to be examined simultaneously at one time for their intrinsic progenitor colony forming ability. This method can help answer questions regarding the biology of pancreatic stem and progenitor cells, such as which markers do progenitor cells express, and which intrinsic or extrinsic factors are important for the maintenance self-renew and differentiation of these cells. The implications of this technique can extend towards the therapy of type one diabetes because if the pancreatic progenitor cells can be identified and differentiated towards beta like cells, then those cells can be used to replace damaged beta cells in patients that have end stage type one diabetes.

It can also be applied to other systems such as studying the colony, forming progenitor cells of pan postnatal and adult pancreas, bone marrow, liver, and spleen. Generally, individuals new to this technique will struggle because dissociating the cells into a single cell suspension and handling the matrigel and methylcellulose in preparation for dispensing can be difficult. I first had the idea for this method when I recognized there was no clon genic assay to address the lineage relationship between progenitor cells and their differentiate progeny from the pancreas.

Such a colony assay has been instrumental for studying hematopoietic stem cell and progenic cells in the past. I recognize a need for such an assay for studying pancreatic stem cell biology. Visual demonstration of this method is critical because the semi-solid median component is viscous by nature and the preparation and dispensing steps are difficult to learn.

Demonstrating the procedures will be Mr.Michael Winkler, a master student, miss Nancy, true a technician, and Dr.Lang Jing, a postop fellow from a lab To generate conditioned medium for the colony assay that will be performed later. Neuron ES cells are differentiated into pancreatic like cells in vitro. Begin this procedure by examining six day old embryo bodies under a light microscope.

At least 30 to 40%of them should have diameters of at least 150 microns. The larger embryo body should contain dark spots suggesting enhanced differentiation. Transfer 80 to 100 embryo bodies to each well of a six Well attachment plate then gently swell the plate to gather the embryo bodies into the centers of the wells to increase cell to cell contacts.

These contacts result in better development of pancreatic like cells from embryo bodies. Gently place the plates back in the incubator at 37 degrees Celsius and 5%carbon dioxide on culture. A 13.

Replace the medium with fresh attachment culture medium containing nicotinamide excend in four and human recombinant active in beta B to induce endocrine cell differentiation. Then return the cultures to the incubator on culture. Day 16, collect the medium and filter it through a 0.2 micron polyethers sulfate membrane to get rid of any cell debris.

From this point forward, the collected medium will be referred to as conditioned medium aliquot the conditioned media into 15 milliliter conical polypropylene tubes and store them at minus 80 degrees Celsius until just prior to use neurogen in three is a transcription factor expressed by pancreatic endocrine progenitors during development. Endocrine precursors expressing neurogen in three ultimately differentiate into pancreatic alpha, beta delta epsilon, and pancreatic polypeptide expressing cells to facilitate the identification of the cells expressing NG N three one allele of the NNG N three locus was replaced with the EGFP reporter gene in a knockin murine ES cell line. The resulting cell line was designated as NGN three EGFP to obtain differentiated NGN three EGFP positive and NGN three EEG FP negative cells.

The cells are first attached from the culture plates by trypsin on day 16 to produce a single cell suspension. The cell clumps are incubated with collagenase B and DNAs one and pass through a 40 or 70 micron mesh. Next DPI is added and the cells are sorted on a cell sorter to sort forward versus side scatter, which are indicative of cell size and granularity respectively are plotted.

A gate is then drawn to eliminate non cellular debris. DPI is used to discriminate dead cells, which will be stained positive. When excited at 4 0 5 to four 13 nanometers and emission detected with a 4 54 40 filter gating of 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 gating of EGFP channel FL one versus autofluorescence.

Using day 16 cells from a control parental cell line, such as TL one shown in the left panel illuminates true fluorescence and thus the NGN three EEG FP positive cell population can be visualized as seen in the right panel. The vinyl sort gate of NGN three EGFP positive cells is intentionally moved to the right as indicated here by the arrow in order to limit contamination of NG N three EEG FP negative cells during sorting. Once the gates have been established, the NGN three EEG, FP positive and NGN three EGFP negative cells are sorted and stored on ice until use in preparation for plating.

Place the matri gel on ice at four degrees Celsius the night before the experiment. On the day of the experiment though, the following reagents or solutions on ice D-M-E-M-F 12 methyl cellulose conditioned media FCS nicotinamide XTEND in four VEGF A and recombinant human active in beta B place a sterile box of 200 microliter pipette tips at minus 20 degrees Celsius at least half an hour before use In preparation to plate the sorted cells, the semi-solid media components must first be added to a five milliliter polystyrene tube. First, use a 16 and a half gauge needle to draw a small amount of 3.3%methyl cellose solution into a syringe.

Then immediately push the solution out to expel the air. This will fill the dead space in the syringe with solution and facilitate a correct measurement of the volume of semi-solid solution. Then avoiding the formation of air bubbles, draw and dispense the intended volume of methyl cellose solution into a five milliliter tube.

Add the conditioned medium FCS nicotinamide extending four active in beta B, VEGF A and D-M-E-M-F 12. Then use the cold pipette tips to add the matrigel. Lastly, add the desired amount of sorted cells into the five milliliter tube.

Always add the cells last to avoid premature stimulation of the cells. Then firmly cap the tube and shake the tubes vigorously and thoroughly by hand. Since shaking generates air bubbles, place the tubes on ice for five minutes or until the small air bubbles surface.

Using a one milliliter syringe with an 18 and a half gauge needle draw in a small amount of the semi-solid media cell mixture and immediately expel the air in the syringe. Then slowly dispense 500 microliters of the semi-solid cell mixture directly into the center of each well of a 24 well low attachment plate. Each sample should be plated in quad duplicate only the four inner the most columns of the horizontally oriented 24 well plate are used for culture.

Fill the remaining wells with sterile water to aid humidification of the cell containing culture. Immediately after plating. View the cells under a light microscope.

Single cells are randomly and evenly distributed throughout the wells. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide air for eight to 12 days. After incubating for eight to 12 days, observe the cells again under a microscope.

This is a good example of evenly distributed matrigel in culture. Here is an example of unevenly distributed matri gel resulting in Apache framework. Colonies do not grow where matri gel is absent.

This is an example of the background of the well where no matri gel was added to the semi-solid media mixture. To count the resultant colonies attach a grid under the plate. Then using a 10 times objective lens, observe and count the colonies due to the three dimensional nature of the medium, 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 representative photo micrographs of insulin expressing colonies in semi-solid culture after eight to 12 days are shown here, colonies indicated by the arrows are randomly distributed and appear a small, dark, non light reflective clusters with a diameter of approximately 60 to 100 microns. Subsequent microfluidic, R-T-P-C-R analysis of individually handpicked colonies revealed expression of insulin two and glucagon consistent to our previous report. These results demonstrate the ability of single NGN three EGFP positive cells to differentiate into colonies containing two eyelet hormones simultaneously resembling primitive first wave-like endocrine cells, protein expression of eptide, a marker for de novo insulin production was confirmed using whole mount fluorescent staining.

In summary, when the insulin expressing colony forming units assay is performed as illustrated in this report, the number of individual progenitors that have the capability to form colonies can be readily determined as demonstrated here and in our previous report, insulin expressing colony forming units can be enriched by sorting with NGN EGFP positive, but not ngn three EEG FP negative cells derived from rine ES cells in vitro Once mastered, this technique can be done in one to three hours. After sorting While attempting this procedure, it's important to remember to completely dissociate the cells into a single cell suspension and keep all the culture components on ice to avoid the solidification of matrigel and add the cells to the tubes last to avoid premature stimulation of the cells Following this procedure. Other methods such as single cell manipulation can be performed to answer additional questions such as whether a single cell is sufficient to initiate colony formations After its development.

This technique will pave the way for the researchers in the field of pancreatic stem and progenic cell biology. It allows them to explore the self-renewal capacity and differentiation capability towards beta like cells. After watching this video, you should have a good understanding of how to conduct the quantitative colony assay for insulin expressing colony forming progenitors.