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Tips for Efficient Generation of hPSC-Derived MPCs
Tips for Efficient Generation of hPSC-Derived MPCs
Human pluripotent stem cell (hPSC)-derived mesenchymal progenitor cells share many of the same characteristics as primary mesenchymal stromal cells (MSCs). Consequently, hPSC-derived MPCs are a popular tool for MSC-related drug discovery and disease modeling, both as an intermediary cell type and as a target for research.
In this Tech Tip, we provide solutions to challenges that have been encountered by scientists when generating MPCs from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). To get the highest quality cells, it is essential to control the differentiation process, which is why we created our optimized differentiation protocol and timeline (Figure 1). For specific workflow details, please refer to the Product Information Sheets (PISs) for the products referenced below (PISs may be found on the respective product webpage).
Figure 1. Optimized Protocol for Differentiating Human Pluripotent Stem Cells into Functional Mesenchymal Progenitor Cells
| Problem Observed | Potential Causes | Recommended Solutions |
|---|---|---|
| Phase 1: Passaging hPSCs for Mesenchymal Progenitor Induction | ||
| Poor mesenchymal induction; for example, early mesoderm markers such as Brachyury are not expressed by Day 4 | Starting hPSCs were of poor quality. | This protocol is compatible with hPSCs maintained in mTeSR™1, mTeSR™ Plus, and TeSR™-E8™, which are then dissociated as single cells into STEMdiff™-ACF Mesenchymal Induction Medium to initiate the generation of MPCs. When transitioning cells from other maintenance media, passage cells a couple of times into the above-tested media before starting mesoderm induction and MPC generation. Follow the tips below to assess and ensure the quality of your starting cells:
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| Starting hPSCs were plated at suboptimal density on Day -2 | The recommended cell density is 5 x 10^4 cells/cm2 when hPSCs are seeded as single cells on Day -2. This should achieve ~30 - 50% confluency on Day -1. If needed, adjust cell density to achieve this confluency on Day -1. The optimal seeding density can also vary between starting hPSC cell lines, so try at least two seeding densities to optimize the protocol for your experiment and starting cells. On Day 0, when the medium is switched to STEMdiff™-ACF Mesenchymal Induction Medium, the cells should be at ~45 - 65% confluency. See Figure 2. |
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| Phase 2: Generation of Early Mesodermal Progenitor Cells | ||
| Medium becoming yellow quickly Many dead and floating cells observed in culture |
Using less than 3 mL of STEMdiff™-ACF Mesenchymal Induction Medium | The PIS states to add 3 mL of the Induction Medium per well (for a 6-well plate). Adjust volumes when using other size cultureware accordingly. Using at least 3 mL is critical, as this is the selection/induction stage where many cells will be lost/detached from the culture. This volume ensures sufficient nutrients for the cells. The 3 mL volume is also important during selection, where there will be many dead and floating cells. The dead, floating cells in 2 mL of supernatant are more concentrated (than in 3 mL), which results in the medium becoming yellow faster. Consequently, using less than 3 mL of media may cause suboptimal induction (due to the release of chemicals from dead cells or other unknown causes), particularly for specific cell lines. |
| Poor mesenchymal induction or excessive peeling by Day 4 of protocol | Refer to recommendations from Phase 1 above. | |
| Phase 3: Derivation of Mesenchymal Progenitor Cells | ||
| Debris in complete MesenCult™-ACF Plus Medium (Figure 3) | Medium stored improperly | Follow the PIS when storing and using the medium. Ensure that the complete MesenCult™-ACF Plus Medium is used within 1 week of preparation and that it is not frozen as aliquots. |
| Natural phenomenon during early passages (cell line-dependent) | Some starting hPSC cell lines are more sensitive than others. It has been observed that P2 is when the cells are typically the most sensitive and there can be a lot of cell death and slow growth. This issue is usually resolved over the next few passages as the cells establish the MPC phenotype. See Figure 3. Daily half-medium changes are only necessary at P1. For P2 or higher, there is no need to change media daily. Half-medium changes are only required if cells start to detach. |
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| Cells failed to reattach after passaging or are detaching from cultureware after being in culture for a couple of days | Did not use Animal Component-Free (ACF) Cell Attachment Substrate or Substrate was not prepared properly. |
When culturing PSC-derived MPCs in MesenCult™-ACF Plus Medium, always use the ACF Cell Attachment Substrate at the correct dilution (1:300 instead of 1:150). Always use D-PBS (Without Ca++ and Mg++) for diluting the Substrate. |
| Use of dissociation reagents (e.g. Trypsin, TrypLE) other than Animal Component-Free (ACF) Cell Dissociation Kit. | Switch to the ACF Cell Dissociation Kit, and use the ACF Enzyme Inhibition Solution to stop the reaction rather than diluting out with MesenCult™-ACF Plus Medium. hPSC-derived MPCs and primary MSCs cultured in MesenCult™-ACF Plus Medium and dissociated with other reagents are more sensitive and exhibit slower expansion passage over passage than those dissociated with the ACF Cell Dissociation kit. This is because dissociation reagents like TrypLE or Trypsin require quenching to stop the reaction; however, as MesenCult™-ACF Plus Medium is free of animal components, it does not contain enough protein (being ACF, serum free) to quench the dissociation reaction. As a result, dilution of dissociation reagents with MesenCult™-ACF Plus Medium will not stop the dissociation reaction, which may cause the digestion of adherence molecules required for the cells to reattach to the cultureware in the next passage. |
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| Cells proliferating slowly in MesenCult™-ACF Plus Medium and on Animal Component-Free (ACF) Cell Attachment Substrate Cultures entering into senescence at early passages Cultures exhibit low MSC markers such as CD90 |
Did not passage when the culture is at 80% confluence (Figure 4) | It is crucial to passage cells when cultures are at 80% confluency (usually takes 3 - 6 days in culture depending on cell line and seeding density). Passaging over-confluent cultures can result in cell-to-cell contact inhibition, which leads to slow cell proliferation. Passaging over-confluent cultures result in cells with poor phenotype (e.g. low CD90 expression). Passaging MPCs when they are under-confluent can also lead to lower cell expansion in subsequent passages. |
| Medium was expired or prepared improperly. | When preparing complete MesenCult™-ACF Plus Medium, always supplement with 2 mM L-glutamine (do not use above or below this concentration). | |
| Cells sticking to tubes after centrifugation | Incompatible plastic tubes used for collecting cells. | Use polypropylene tubes to prevent cells from sticking. |
| Cells are not evenly distributed in the well. They are more confluent in the center of the well and less so around the edges | Cells were not mixed properly following seeding | To achieve a homogeneous cell distribution, immediately after seeding, move the cultureware in several quick, short back-and-forth and side-to-side motions to evenly distribute the MPCs across the surface. Then, place the cultureware in a 37°C incubator and repeat these same quick, short motions to ensure even distribution. Because MPCs adhere rapidly to the cultureware, it is essential not to skip this step. Repeating the motion in the incubator is also important, as carrying the cultureware from the microscope or biosafety cabinet to the incubator can cause the cells to pool in the center of the well. |
Figure 2. Day 0 Cell Confluency
Representative images of cell confluency at Day 0 of the STEMdiff™ Mesenchymal Progenitor Kit protocol from human ESC (H9) and iPSC (3A, 4A) cell lines. Note that the optimal seeding density at Day -2 can vary between cell lines and it is recommended that customers seed at least 2 densities (e.g. 2 x 10^4 cells/cm2 and 5 x 10^4 cells/cm2 for each condition) to optimize this stage for their experiments. These images are on the lower end of an ideal confluency and were imaged in the lower seeded wells of this experiment.
Figure 3. Cell Debris in Culture at Passage 2
This figure shows two passages from the same culture (Passage 2 and 6). Both passages are at the same time point (3 days after seeding) and both use the same seeding density. Passage 2 has been observed to have slower cell expansion and more cell death as seen here with more floating dead cells and debris in culture (indicated with white arrow). Despite this, cultures are observed to recover over the next few passages as cell expansion picks up and cell death decreases, as seen in the Passage 6 image.
Figure 4. Optimal MPC Confluency for Passaging
MPCs grow optimally when they are passaged at ~80% confluency. Here are some representative images of the lowest, ideal, and highest confluency that we would recommend for passaging. Passaging under- or over-confluent MPC cultures can have a negative effect on subsequent passages, with poor morphology and cell expansion observed.
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