TITLE: Reference pictures of hPSC cultured in defined conditions
AUTHORS: Katarzyna Ludwik, Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
Collection of reference images of hPSCs in different culture conditions and at different timepoints. 

[STEPS]
SECTION: Example images of undifferentiated hPSCs in different culture conditions
1. Timepoints, matrix, and media used are labeled or indicated in Step description. 

The standard morphology of undifferentiated hPSC colonies is distinctive and serves as an important criterion for assessing the pluripotent state of the cells. Here is a detailed description:

Shape and Edges:
Colonies are typically round or oval with well-defined, smooth edges.
Edges should be tight and compact, without cells protruding or migrating outward (which suggests differentiation or unhealthy colonies).

Cell Density and Arrangement:
Colonies appear densely packed, with little or no visible space between cells.
The central area of the colony is more densely populated, with cells tightly connected via intercellular junctions.

Cell Appearance:
Cells within the colony are generally small, with a high nucleus-to-cytoplasm ratio.
Nuclei are large, round, and centrally located, often with prominent nucleoli visible under phase-contrast or bright-field microscopy.
The cytoplasm is scant and difficult to distinguish.

Color and Texture (in phase contrast):
Colonies have a refractive (bright) edge under phase contrast, indicating tight cell-cell contact.
The center may appear slightly raised or dome-shaped due to the dense packing of cells.

SECTION: Example images of undifferentiated hPSCs in different culture conditions
1.2. Additional examples of healthy hPSC colony morphology. 
SECTION: Example images of spontaneous differentiation of hPSCs
4.1. Examaples of spontaneous differentiation of hPSCs. 
 

SECTION: Example images of spontaneous differentiation of hPSCs
4. Description of spontaneous differentiation of hPSCs.

Spontaneous differentiation of pluripotent stem cells is a common phenomenon that can occur in standard culture conditions, especially when colonies are overgrown, improperly maintained, or exposed to suboptimal media or environmental stress. Morphological cues are often the earliest indicators of this unwanted differentiation. Below is a detailed description of the characteristics and patterns of spontaneous differentiation in hPSC cultures:

Irregular, Flattened Cells:
Differentiating cells at the colony periphery often become flattened, spread out, and less refractile.
These cells lose the tight packing and cobblestone-like morphology typical of undifferentiated iPSCs.
There is a visible loss of junctional integrity, leading to gaps between cells or protrusions extending from the colony edge.
A mixed population of cells may emerge, with some appearing epithelial-like, while others may exhibit mesenchymal traits, depending on the differentiation trajectory.

Raised, Dome-Like Areas:
Overgrown colonies often develop elevated central mounds or domes, which may be indicative of spontaneous differentiation initiation.
These dome regions may contain a mix of larger cells with abundant cytoplasm and cells forming rosette-like or pseudo-epithelial structures, suggesting early lineage commitment.
The architecture within domes often shows loss of planar polarity, with cells oriented in various directions.

Color and Texture Variations:
Differentiating regions appear less shiny and more opaque under phase-contrast microscopy.
Colonies lose their uniform refractive edge and develop a "ruffled" or scalloped appearance.
When subcultured, differentiated cells often fail to re-establish tight colonies and instead grow as dispersed monolayers.
SECTION: Example images of undifferentiated hPSCs cultured with survival factors
3. Timepoints, matrix, and media used are labeled or indicated in Step description. 

When hPSC are cultured in media supplemented with a survival factors, their appearance undergoes notable changes, especially during the early phases after passaging. These are used to enhance cell survival—especially after enzymatic dissociation into single cells—and reduce apoptosis.
 
Immediate Post-Passage (0–24 hours)

Cell Shape:
Cells tend to be rounder and more spherical compared to their flattened counterparts in non-ROCK conditions.
These cells are highly refractile and may look less adherent initially.
Cytoplasmic processes or spreading are minimal during this phase.

24–48 Hours Post-Passage
Colony Initiation:
Cells begin forming tight, cohesive colonies, resembling compact epithelial-like clusters.
The cells become more flattened and start displaying typical hPSC morphology as they re-establish contacts.
The classical appearance of hPSCs with large, prominent nuclei and scant cytoplasm re-emerges.
Nuclei remain round and centralized, with visible nucleoli under high magnification.

Longer-Term Cultures (48+ hours)
Colonies regain typical undifferentiated morphology, as described in your previous section.
Survival factors are  typically removed after 24–48 hours.
SECTION: Example images of undifferentiated hPSCs in different culture conditions
1.1. Morphology comparison for hiPSC cells cultured on either Geltrex or Vitronectin coated plates. 
 

 

 

 

 

 

SECTION: Example images of undifferentiated hPSCs cultured with survival factors
3.2. Example images of undifferentiated hiPSC lines one day after passaging with TrypLE Select and plated on Geltrex. Note the single cell morphology due to presence of ROCK inhibitor. This morphology disappears after media change to media without ROCK inhibitor. See examples in the next step. 
 

SECTION: Example images of undifferentiated hPSCs cultured with survival factors
3.3. Example images of morphology changes after single cell passaging of hPSCs, plating in media supplemented with indicated survival factors (ROCK inhibitor or CEPT), which were subsequently removed after 24h. 
 

 

 

SECTION: Example images of undifferentiated hPSCs cultured with survival factors
3.4. Example images of morphology changes after single cell passaging of hPSCs and plating in media supplemented with CloneR2.
 

SECTION: Example images of undifferentiated hPSCs cultured with survival factors
3.1. Example images of undifferentiated hiPSC line after thawing and after first passage. Note the single cell morphology (due to ROCK inhibitor) and presence of floating cells in the culture 1 day after thawing. Cells show expected colony formation after 5 days in culture. 
 

SECTION: Example images of undifferentiated hPSCs in different culture conditions
1.3. Additional examples of healthy hiPSC colony morphology accuired with Oblique Coherent Contrast (OCC) Illumination (Stereo microscope). 
 

SECTION: Example images of spontaneous differentiation of hPSCs
4.2. Additional examples of hiPSC differentiation acquired with Oblique Coherent Contrast (OCC) Illumination. 
 

SECTION: Example images of undifferentiated hPSCs in different culture conditions
2. Confluency assessment

Evaluating confluency of hPSC cultures is essential for monitoring growth, timing passaging, and ensuring reproducibility in downstream applications. 
Confluency refers to the percentage of the surface area of a culture vessel (e.g., dish or flask) covered by adherent cells. For hPSCs, this reflects the extent to which colonies occupy the culture surface.

How to Evaluate Confluency:

Visual Estimation
Confluency is estimated by looking at the culture under a microscope and judging what percentage of the surface is covered by cells. This method is quick and requires no equipment beyond the microscope, but it is subjective and depends on the observer’s experience.

Image Analysis (Software-Based)
A more objective approach involves taking images of the culture and analyzing them using a software. This process typically includes converting the image to grayscale, applying a threshold to separate cells from the background, and calculating the area percentage covered by cells. Tools like ImageJ, CellProfiler, or custom Python scripts are well suited for this method and offer more consistent, reproducible results. This method is time consuming and requires image analysis expertise.

Live-Cell Imaging Systems
Automated systems such as IncuCyte or BioStation can track confluency in real time by capturing images over time inside an incubator and estimating confluency using built-in image analysis pipelines. These systems are ideal for long-term studies or high-throughput applications because they reduce manual work and provide continuous, standardized data on culture growth. This method requires specialized equipment. 
SECTION: Example images of undifferentiated hPSCs in different culture conditions
2.1. Example images of healthy hiPSC colonies acquired with Oblique Coherent Contrast (OCC) Illumination reflecting various confluency levels. 
 

SECTION: Example images of undifferentiated hPSCs in different culture conditions
2.2. Example images of healthy hiPSC colonies acquired with automated imaging system IncuCyte reflecting various confluency levels over time.