A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described. 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 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 β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.
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 — a so called “black box” 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 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 β 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 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.
Notes before getting started:
1. Dissection of Human Fetal Pancreas
2. Immunofluorescence of Markers of Endocrine Cell Proliferation and Maturation in ICCs Grown in Suspension, Monolayer, or on Glass Coverslips
3. (Optional) Immunofluorescent Staining of Proteins from Paraffin Embedded ICCs
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-μm 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).
Figure 1. Flow chart of human fetal ICC preparation and staining for Immunofluorescence.
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 µm, scale bar for D, 20 magnification = 100 µm. Please click here to view a larger version of this figure.
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 μm.
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 β 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 β 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.
The authors have nothing to disclose.
This work was supported by the California Institute for Regenerative Medicine (RB3-02266) and the NIH (DK54441).
Trehalose SG | Hayashibara | Trehalose SG | |
Collagenase XI | Sigma | C-9407 | |
Hank’s Balanced Salt Solution (HBSS) | Life Technologies | 24020-117 | |
RPMI 1640 with glutamate | Life Technologies | 11879020 | no glucose or HEPES |
Human AB Sera, Male Donors | Omega Scientific | HS-30 | |
Fungizone | Life Technologies | 15290018 | |
Gentamicin Solution 50 mg/ml | Invitrogen | 15750060 | |
1M Hepes, pH 7.0 | Life Technologies | 15630080 | |
Penicillin – Streptomycin 100X Solution | Life Technologies | 15070063 | |
Glutamax | Life Technologies | 35050061 | |
Hepatocyte Growth Factor (HGF) | Peprotech | 100-39 | |
GLP-1 | Peprotech | 130-08 | |
Fetal Bovine Serum | Invitrogen | 16000-044 | |
Dnase | Sigma | DN25 | |
Sterile Petri Dishes, 60 x 15 mm; Stackable, venting ribs | Spectrum Laboratory Products, Inc. | D210-13 | |
PBS | Gibco | 14190 | |
16% PFA | Electron Microscopy Sciences | 15710 | |
Triton X-100 | Sigma | T9284 | |
Donkey Serum | Jackson ImmunoResearch | 017-000-121 | |
BrdU | Life Technologies | 00-0103 | |
anti-insulin (mouse monclonal; 1:1000) | Sigma | I2018 | |
anti-glucagon (mouse monoclonal; 1:2000) | Sigma | G2654 | |
PDX1 (mouse monoclonal) | Novus Biologicals | NBP1-47910 | |
PDX1 (goat polyclonal) | AbCam | 47383 | |
PDX1 (rabbit polyclonal) | AbCam | 47267 | |
AlexaFluor 546 (rabbit) | Invitrogen | A11010 | |
AlexaFluor 546 (mouse) | Invitrogen | A11003 | |
AlexaFluor 488 (rabbit) | Invitrogen | A11008 | |
AlexaFluor 488 (mouse) | Invitrogen | A11001 | |
Ki67 | Lab Vision Neomarkers | RM 9016-50 | |
pan-CK (mouse monoclonal; 1:100) | Immunotech | 2128 | |
CK19 | AbD Serotech | MCA 2145 | |
DAPI | Cell Signaling | 4083 | |
HTB9 cell line | ATCC | 5637 | see Beattie 1997 reference to generate matrix |
Agarose | Sigma | A-6013 |