Lesson 8 Hillreiner et al., 2017

Applied Molecular Cellular Biology

🧫 Why This Study?

Researchers wanted a better model to study how cow mammary cells produce milk and respond to infection. Most labs use cell lines or biopsy-derived cells, but those don’t behave like real mammary tissue. So they used primary bovine mammary epithelial cells (pbMEC) — the milk-producing cells — that they non-invasively collected from fresh milk. 🥛 These cells were grown in a 3D “mini-udder” model on Matrigel®, a gel mimicking the extracellular matrix (ECM).

🧬 Experimental Setup

Milk collection: From healthy Brown Swiss cows.
Cell isolation: pbMECs were separated from 1 L of milk and cleaned through filters (100 μm → 40 μm).
Cultures:
- 2D control: cells on flat plastic.
- 3D culture: cells embedded in Matrigel®.
Media contained DMEM F12, hydrocortisone, and ITS (insulin-transferrin-selenite).
Treatments tested:
- - Prolactin (PRL) 🧴
- - L-lysine (LYS) 🍖
- - Both
Cells grew for 4 days; media were collected daily for analysis.

🔬 Techniques Used

Immunocytochemistry → verified epithelial identity using cytokeratin antibodies.
Microscopy & viability dyes → showed living, proliferating “mammospheres” forming in 3D.
RT-qPCR → measured expression of:
- Milk proteins (CSN1S1, CSN2, CSN3).
- JAK-STAT pathway genes (STAT5A, PRLR, CEBPB, etc.).
- mTOR pathway genes (RPS6KB1, AKT1, EIF4EBP1).
Proteomics (LC-MS/MS) → identified secreted proteins in the culture medium (“secretome”).

🧩 Key Results

🧁 1. 3D Cultures = Realistic Mini-Alveoli

Cells on Matrigel® formed alveolar-like spheres, mimicking milk-producing units in the udder. They were alive, polarized, and functional — unlike flat 2D monolayers.

🍼 2. Milk Protein Gene Expression

αS1-casein (CSN1S1): strongly induced in 3D and by PRL or LYS.
β- and κ-caseins: also up, but more variable.
ECM contact was crucial — matching previous work showing basement membrane signals activate milk genes.

⚙️ 3. JAK-STAT Pathway

This hormone-signaling pathway controls lactation.
3D culture upregulated STAT5A, CEBPB, and YY1.
PRL + LYS combo actually lowered YY1 (a transcription repressor) → promoting milk gene expression.

💪 4. mTOR Pathway

mTOR controls protein synthesis and cell growth.
RPS6KB1 and AKT1 were significantly upregulated in 3D, linking ECM to enhanced biosynthesis.
The 3D environment had a stronger effect than hormones.

💧 5. Secreted Proteins (Proteomics)

Detected 56 proteins 🧫:

Milk & whey proteins: αS1-casein, β-casein, α-lactalbumin, β-lactoglobulin.
Proliferation/differentiation: 14-3-3σ (SFN), TGFB1, galectin-3.
Cytoskeleton/structure: actinin-1, cofilin-1, calponin-2.
Immune factors: lactoferrin (LTF), β2-microglobulin (B2M), chemokines CXCL3 & CXCL6.

✅ This shows pbMECs in 3D are metabolically active, polarized, and immunocompetent — like real mammary tissue.

🧠 Discussion Highlights

Milk-cell extraction is ethical, reproducible, and avoids fibroblast contamination.
3D culture preserves natural polarity and cell-cell communication, lost in 2D.
The ECM scaffold (Matrigel®) drives lactation genes more than hormones alone.
pbMECs in 3D secrete immune molecules, meaning this model can study both lactation and mastitis defense.

🎯 Conclusion

Hillreiner et al. created a functional 3D model of cow mammary epithelium directly from milk — ✅ non-invasive ✅ physiologically realistic ✅ usable for studying milk production, hormone signaling, immune defense, and metabolism

This “mini-udder in a dish” 🌸 opens the way for future studies on lactation, mastitis, and metabolic disorders — all without harming the animal.

Quiz

Score: 0/30 (0%)

Q0. What was the main goal of Hillreiner et al. (2017)?

To test antibiotics against mastitis pathogens

To establish a non-invasive 3D culture model of bovine mammary epithelial cells from milk

To improve milk yield in dairy cows

To compare milk composition among different cow breeds

Q1. Why did the authors choose cells isolated from milk instead of udder biopsies?

Milk cells are easier to stain for cytokeratin

Isolation from milk is non-invasive and avoids fibroblast contamination

Milk cells grow faster than biopsy cells

Milk samples contain more immune cells

Q2. Which scaffold was used to mimic the extracellular matrix in the 3D culture?

Collagen I

Laminin gel

Matrigel®

Fibronectin film

Q3. What key feature did pbMECs form when grown in 3D on Matrigel®?

Monolayers of flat cells

Alveolar-like mammospheres

Fibroblast-like sheets

Multinucleated syncytia

Q4. Which hormone was used to stimulate milk protein gene expression?

Estrogen

Cortisol

Prolactin

Progesterone

Q5. What was the purpose of including L-lysine in some treatments?

To test its role in mTOR-mediated protein synthesis

To replace glucose as a carbon source

To promote apoptosis

To inhibit cytokine expression

Q6. Which method did the researchers use to analyze secreted proteins?

ELISA

LC-MS/MS

Western blot

Chromatography only

Q7. Which gene showed a statistically significant induction in 3D culture compared to 2D?

CEBPB

PRLR

JAK2

RUNX2

Q8. What did 'a differential gene expression for AKT1' indicate?

AKT1 was inactive in all treatments

AKT1 expression changed significantly between 3D treatment conditions

AKT1 was silenced in the 2D culture

AKT1 was unaffected by culture conditions

Q9. Which group of proteins was NOT among those identified in the secretome?

Milk and whey proteins

Cytoskeletal organization proteins

Viral capsid proteins

Immune response proteins

Q10. Which statement best describes fibroblasts in this context?

They are desired for milk protein secretion

They contaminate mammary cell cultures

They are part of the alveolar epithelial layer

They are immune cells in milk

Q11. What was the advantage of using primary cells over stable cell lines?

They divide faster

They preserve in vivo-like physiology

They are easier to genetically modify

They require fewer nutrients

Q12. Which two signaling pathways did the authors focus on?

Notch and Hedgehog

MAPK and TGF-beta

JAK-STAT and mTOR

Wnt and NF-kB

Q13. What is meant by a 'tissue-specific polarized phenotype' in 3D culture?

Cells lose their differentiation markers

Cells display defined apical and basal sides like real tissue

Cells become spherical and undifferentiated

Cells align randomly without structure

Q14. Which statement about YY1 gene expression is true?

It was downregulated by PRL + LYS treatment compared to control

It was upregulated by all treatments

It was not expressed in pbMEC

It only appears in fibroblasts

Q15. 3D culture increased expression of αS1-casein compared to 2D.

True

False

Q16. Matrigel® mainly contains laminin, collagen IV, and growth factors.

True

False

Q17. Prolactin activates milk protein genes via the mTOR pathway.

True

False

Q18. The pbMEC in this study were obtained through invasive surgery.

True

False

Q19. Fibroblast contamination is less likely in milk-derived pbMEC cultures.

True

False

Q20. L-lysine directly inhibits casein gene expression.

True

False

Q21. CEBPB and STAT5A are both transcription factors in the JAK-STAT pathway.

True

False

Q22. The term 'secretome' refers to all RNA molecules expressed in a cell.

True

False

Q23. AKT1 is a component of the mTOR signaling pathway.

True

False

Q24. The presence of chemokines like CXCL3 and CXCL6 indicates immune functionality in pbMEC.

True

False

Q25. RT-qPCR was used to analyze protein content in the medium.

True

False

Q26. 3D culture models better mimic in vivo cell-cell communication than 2D models.

True

False

Q27. Hydrocortisone was used to induce alveolar differentiation in pbMEC.

True

False

Q28. The study confirmed that pbMEC in 3D culture can secrete milk and immune-related proteins.

True

False

Q29. The JAK-STAT pathway is unrelated to lactation.

True

False