**5. Practical applications of the pgMECs**

Mammary development, hormone responsiveness of mammary cells, regulation of milk expression, modeling milk composition and coagulation properties, enhancing milk yield, and innate immunity are some of the interests of modern dairy production. The characterized mammary cell lines are useful models to study biology of the mammary gland. For example, we used the cells for infection study with a common mastitis-causing agent in goats—*Mycoplasma agalactiae* (PG2 strain)—and to study differences in expression of the steroid receptors and beta casein in different growth conditions and in different pgMEC lines, derived from tissues of animals in different physiological states.

#### **5.1. Mastitis model**

antibodies raised against KRT14 and 18. They consisted of several hundred cells (**Figure 6D**), which were KRT14- or KRT18-positive. Hanging drop is an efficient method to grow mammospheres from primary mammary cultures. The cells avoiding anoikis and forming organoids

**Figure 6.** Formation of spherical structures in the pgMECs after 7 days of growth under nonadherent conditions (photo: J. Ogorevc). (A) Spherical structures in low-attachment plates (20× magnification; scale bar = 50 μm). (B) Organoids (mammospheres) grown in hanging drops (4× magnification; scale bar = 200 μm). (C and D) DAPI-stained fixed mammospheres, grown in hanging drops under bright field (C) and fluorescent illumination (D) (100× magnification; scale bar = 100 μm). Number of cells in the organoid structures can be estimated based on the number of stained nuclei

Mammary development, hormone responsiveness of mammary cells, regulation of milk expression, modeling milk composition and coagulation properties, enhancing milk yield, and innate immunity are some of the interests of modern dairy production. The characterized mammary cell lines are useful models to study biology of the mammary gland. For example, we used the cells for infection study with a common mastitis-causing agent in goats—*Mycoplasma agalactiae* (PG2 strain)—and to study differences in expression of the steroid receptors and beta casein in different growth conditions and in different pgMEC lines,

probably represent mammary progenitors.

(blue).

176 Goat Science

**5. Practical applications of the pgMECs**

derived from tissues of animals in different physiological states.

Because of the economic importance for dairy industry and possible health and milk quality risks for consumers, there is a great interest to understand and enhance natural immunity of the mammary gland. Mammary epithelial cells are capable of innate immune response during intramammary infections and represent important barrier against invading pathogens.

In small ruminants, coagulase-negative staphylococci account for most of the mastitis cases, followed by Streptococci, *Staphylococcus aureus*, and other bacteria [23]. Additionally, contagious agalactia caused by *Mycoplasma agalactiae* (*Ma*) is a common cause of intramammary infections (contagious agalactia), especially in Mediterranean regions [24]. Mammary cell lines are often used to study immune response to mastitis, instead of *in vivo* infections. In our study, next-generation sequencing (NGS) was used to assess whole-transcriptomic response of *Ma*-challenged pgMECs 3, 12, and 24 h postinfection [25].

The results show that the infection induced an innate immune response in the infected cells. The pgMECs were capable of recognizing and responding to the pathogen infection (**Figure 7**). The pgMECs responded by induced expression of cytokines (interleukins and chemokines) and other chemotactic agents, activation of complement system, apoptosis pathways, and induction of genes coding for antimicrobial effector molecules (e.g. defensins, lysozyme, and nitric oxide synthase) (**Figure 7A**). The changes in expression were moderate, with no phenotypically visible changes in cell morphology, which corresponds to subclinical course of contagious agalactia *in vivo*. The pathway enrichment analysis showed that the most affected pathways were associated with immune response, steroid and fatty acid metabolism, apoptosis signaling, transcription regulation, and cell cycle regulation. We speculate that physiologically, the *in vivo* immune contribution of the pgMECs is important for recruitment of

**Figure 7.** Transcriptomic studies on *Mycoplasma agalactiae*–infected pgMECs (modified from Ref. [25]). (A) Induction of immune-associated genes interleukin 8 (IL8), chemokine (C-X-C motif) ligand 5 (CXCL5), Toll-like receptor 2 (TLR2), and S100 calcium-binding protein 9 (S100A9). (B) Possible immune response mechanisms in pgMEC, suggested based on differential expression of genes and analysis of genetic networks and metabolic pathways.

neutrophils, activation of complement system and apoptotic pathways, as well as expression of several bactericidal molecules (**Figure 7B**) [25].

*5.2.2. Expression of the steroid receptors*

Terminal differentiation of mammary epithelial cells is required for luminal cells to secrete milk. Estrogen and progesterone are important hormones in mammary development and mammary cell differentiation. The roles of estrogen and progesterone during morphogenesis are well known [12], but their role during lactation is not clear. The function of the hormones is mediated through estrogen (ESR) and progesterone receptors (PR) that in ligated form migrate to nucleus and act as transcription factors for various genes. The studies suggest that ESR1 and PR are (co)expressed in mammary epithelial cells [30] where they participate in regulation of differentiation and control balance between luminal and basal mammary epithelial cells, mediated through paracrine signaling between the neighboring cells [31, 32]. Most of the ESR1- and PR-positive cells express markers of the luminal lineage [33], whereas the lack of ESR and PR is typical for undifferentiated mammary progenitor cells [34]. Unclear mechanisms by which hormonal action regulates lineage commitment and cell proliferation hamper our understanding of mammary differentiation, potentially useful for boosting milk production, as well as for better understanding of malignant transformations in mammary cells.

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We quantified the expression of the steroid receptors in different pgMEC lines, grown in different conditions [35]. The cell lines derived from the mammary tissue of nonlactating doelings exhibited higher relative expression of ESR1 (approximately 50-fold) and PGR (approximately 8-fold), compared to cells derived from tissue of lactating goats. The response to lactogenic conditions was variable upregulation (from 1.4- to 12-fold) of ESR1 and consistent (approximately 3-fold) downregulation of PGR. Using immunostainings, we identified epithelial cells negative for both receptors, positive solely for ER-α or PR, and cells coexpressing both receptors. ER-α and PR were mainly localized in the nuclei and partly in cytoplasm of the cells. Multiple staining with luminal (CK18) or basal (CK14) markers revealed that not all of the ER-α–positive or PR-positive markers belonged to the luminal lineage and that not all of the luminal cells are ER-α and/or PR-positive (**Figure 9**). It seems that lactogenic conditions caused differentiation and proliferation of the luminal lineage and that ER-α could be

**Figure 9.** Immunofluorescent double stainings of the pgMEC lines, under 200× magnification (photo: J. Ogorevc). Nuclei were counterstained with DAPI (blue). (A) ER-α (green) localized mainly in nuclei of CK18-positive (red) cells. CK18 negative cells with nuclear staining against ER-α can also be observed. (B) PR-positive (green) and PR-negative luminal (red—CK18-positive) cells. (C) Nuclear colocalization of ER-α and PR (orange) and cells positive solely for ER-α (green)

involved in functional differentiation of the luminal mammary cells.

or PR (red).

#### **5.2. Mammary differentiation and lactation model**

Expression of milk proteins and appearance of milk drops containing lipids are signs of lactogenic differentiation of the mammary cell culture. Lactogenic differentiation is dependent on multiple factors. To determine if the derived pgMECs are capable of lactogenic differentiation, we performed screening for beta-casein expression, the most abundant protein in goat milk, and stained putative milk drops for the presence of lipids, using Oil Red O.

Several pgMEC lines from mammary tissue of animals in different physiological states (different stages of lactation and juvenile goats) were established and grown under different growth conditions (basic and lactating). We observed different morphology, expression of steroid receptors (estrogen and progesterone), and expression of beta casein (CSN2), and tried to evaluate the effect of different growth conditions (medium, growth surface, and cell density) and donor tissue on characteristics of the derived cell lines. It was shown that primary mammary cells rapidly loose expression of steroid receptors [26] and casein [27] when grown in monolayer on plastic, whereas growth on extracellular matrices should provide the basalapical polarization to the epithelial cells, needed for maintaining certain characteristics or to enable proper differentiation and milk component synthesis [28, 29].

#### *5.2.1. Morphology*

Morphology differed between the derived primary cultures established from mammary tissue of goats in different physiological states. Cells of the same primary cell line, grown under different growth conditions, also exhibited morphological differences (**Figure 8**). When cells were grown to confluency in lactogenic medium, dome-like and acini-like structures were formed in pgMECs, derived from the lactating tissue. No such structures appeared in pgMECs derived from the tissue of juvenile goats, grown under the same conditions. However, lumenlike and milk drop–like structures were formed only in pgMECs derived from juvenile goats, grown in lactogenic medium.

**Figure 8.** Morphology of different pgMEC lines in lactogenic conditions (photo: J. Ogorevc). Dome-like (A) and acini-like structures (B) were formed in pgMECs, derived from lactating mammary tissue (40× magnification; scale bars = 200 μm). Vacuoles resembling milk drops (arrow) and lumen-like structures (arrowheads) were formed in pgMECs, derived from juvenile goats (200× magnification; scale bar = 50 μm).

#### *5.2.2. Expression of the steroid receptors*

neutrophils, activation of complement system and apoptotic pathways, as well as expression

Expression of milk proteins and appearance of milk drops containing lipids are signs of lactogenic differentiation of the mammary cell culture. Lactogenic differentiation is dependent on multiple factors. To determine if the derived pgMECs are capable of lactogenic differentiation, we performed screening for beta-casein expression, the most abundant protein in goat

Several pgMEC lines from mammary tissue of animals in different physiological states (different stages of lactation and juvenile goats) were established and grown under different growth conditions (basic and lactating). We observed different morphology, expression of steroid receptors (estrogen and progesterone), and expression of beta casein (CSN2), and tried to evaluate the effect of different growth conditions (medium, growth surface, and cell density) and donor tissue on characteristics of the derived cell lines. It was shown that primary mammary cells rapidly loose expression of steroid receptors [26] and casein [27] when grown in monolayer on plastic, whereas growth on extracellular matrices should provide the basalapical polarization to the epithelial cells, needed for maintaining certain characteristics or to

Morphology differed between the derived primary cultures established from mammary tissue of goats in different physiological states. Cells of the same primary cell line, grown under different growth conditions, also exhibited morphological differences (**Figure 8**). When cells were grown to confluency in lactogenic medium, dome-like and acini-like structures were formed in pgMECs, derived from the lactating tissue. No such structures appeared in pgMECs derived from the tissue of juvenile goats, grown under the same conditions. However, lumenlike and milk drop–like structures were formed only in pgMECs derived from juvenile goats,

**Figure 8.** Morphology of different pgMEC lines in lactogenic conditions (photo: J. Ogorevc). Dome-like (A) and acini-like structures (B) were formed in pgMECs, derived from lactating mammary tissue (40× magnification; scale bars = 200 μm). Vacuoles resembling milk drops (arrow) and lumen-like structures (arrowheads) were formed in pgMECs, derived from

milk, and stained putative milk drops for the presence of lipids, using Oil Red O.

enable proper differentiation and milk component synthesis [28, 29].

*5.2.1. Morphology*

178 Goat Science

grown in lactogenic medium.

juvenile goats (200× magnification; scale bar = 50 μm).

of several bactericidal molecules (**Figure 7B**) [25].

**5.2. Mammary differentiation and lactation model**

Terminal differentiation of mammary epithelial cells is required for luminal cells to secrete milk. Estrogen and progesterone are important hormones in mammary development and mammary cell differentiation. The roles of estrogen and progesterone during morphogenesis are well known [12], but their role during lactation is not clear. The function of the hormones is mediated through estrogen (ESR) and progesterone receptors (PR) that in ligated form migrate to nucleus and act as transcription factors for various genes. The studies suggest that ESR1 and PR are (co)expressed in mammary epithelial cells [30] where they participate in regulation of differentiation and control balance between luminal and basal mammary epithelial cells, mediated through paracrine signaling between the neighboring cells [31, 32]. Most of the ESR1- and PR-positive cells express markers of the luminal lineage [33], whereas the lack of ESR and PR is typical for undifferentiated mammary progenitor cells [34]. Unclear mechanisms by which hormonal action regulates lineage commitment and cell proliferation hamper our understanding of mammary differentiation, potentially useful for boosting milk production, as well as for better understanding of malignant transformations in mammary cells.

We quantified the expression of the steroid receptors in different pgMEC lines, grown in different conditions [35]. The cell lines derived from the mammary tissue of nonlactating doelings exhibited higher relative expression of ESR1 (approximately 50-fold) and PGR (approximately 8-fold), compared to cells derived from tissue of lactating goats. The response to lactogenic conditions was variable upregulation (from 1.4- to 12-fold) of ESR1 and consistent (approximately 3-fold) downregulation of PGR. Using immunostainings, we identified epithelial cells negative for both receptors, positive solely for ER-α or PR, and cells coexpressing both receptors. ER-α and PR were mainly localized in the nuclei and partly in cytoplasm of the cells. Multiple staining with luminal (CK18) or basal (CK14) markers revealed that not all of the ER-α–positive or PR-positive markers belonged to the luminal lineage and that not all of the luminal cells are ER-α and/or PR-positive (**Figure 9**). It seems that lactogenic conditions caused differentiation and proliferation of the luminal lineage and that ER-α could be involved in functional differentiation of the luminal mammary cells.

**Figure 9.** Immunofluorescent double stainings of the pgMEC lines, under 200× magnification (photo: J. Ogorevc). Nuclei were counterstained with DAPI (blue). (A) ER-α (green) localized mainly in nuclei of CK18-positive (red) cells. CK18 negative cells with nuclear staining against ER-α can also be observed. (B) PR-positive (green) and PR-negative luminal (red—CK18-positive) cells. (C) Nuclear colocalization of ER-α and PR (orange) and cells positive solely for ER-α (green) or PR (red).

#### *5.2.3. Expression of beta casein (CSN2)*

Expression of caseins was detected in various primary and immortalized mammary cell lines from different species. For example, mouse HC11 cells [36], several bovine mammary cell lines [15, 28, 37–39], and goat primary mammary cells [8, 18, 40] are able to express caseins. Transcription of CSN2 was studied in mouse HC11 cells and the authors [41] found that its expression is induced synergistically by combination of lactogenic hormones, local growth factors, and cell-cell and cell-substratum interactions.

We evaluated how the starting tissue material, addition of hormones (insulin, hydrocortisone, and prolactin) to the growth medium, and growth on a commercially prepared extracellular basement membrane matrix affect relative expression of beta casein [42], determined using RT-qPCR (**Figure 10A**). The CSN2 transcripts were detected in all of the samples, including cells originating from nonlactating goat, grown in basal medium. However, the expression of CSN2 and response to different growth conditions were different in individual cell lines. Interestingly, we found that CSN2 expression was the highest in pgMECs derived from juvenile goats, grown in lactogenic medium. Addition of hormones in most cases induced expression of CSN2. The effect of extracellular membrane matrix–covered growth surface (Geltrex in our case) was variable. We found that extracellular membrane matrix growth surface was not indispensable for casein expression in the cell lines. In some of the cell lines, membrane matrix significantly increased CSN2 expression, whereas in others, it did not have a statistically significant effect (in several cases, expression of CSN2 was even lower [nonsignificantly] in pgMECs grown on membrane matrix). We speculate that some of the cell types present in a heterogeneous mixture of the cell cultures, are capable of extracellular matrix production in quantities sufficient for luminal cells to achieve lactation competency. It was reported previously that mouse HC11 cells can produce extracellular matrix [43] and do not require additional matrix for CSN2 expression [36].

differentiation and proliferation of mammary epithelial cells. Research on humans shows that ER-/PR-positive mammary cells represent early mammary progenies and regulate differentiation of ER-negative mammary progenitors, from which basal/myoepithelial cells arose, and to ER-positive bipotent progenitors, which can give rise to luminal and myoepithelial lineage [33, 44]. It would make sense that progenitor fractions are enriched in juvenile mammary tissue (and in derived pgMECs) where lactogenic differentiation had not occurred yet. Prpar and colleagues [10] reported existence of different mammary progenitors in goats, luminalrestricted, myoepithelial-restricted, and bipotent and showed that the tissue from animals at the peak of lactation and from a juvenile animal contained the highest number of luminal progenitors, whereas the tissue at the onset of involution contained mostly myoepithelial progenitors. In our experience, tissue from young, nonlactating (juvenile) goats seems more suitable for preparation of lactation-competent cell cultures than tissue from lactating or involuting animals. Similar was also suggested in case of bovine primary mammary cells and

**Figure 10.** Expression of CSN2 mRNA and localization of the protein in pgMECs (photo: J. Ogorevc). (A) RT-qPCR amplification plot for housekeeping GAPDH and CSN2 in three different pgMEC lines. (B) Beta casein was present in pgMEC lines and localized mainly in vacuoles (lumen-like structures), formed only under lactation-inducing conditions

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The development of primary cell lines from lactating, juvenile, and involuting goat mammary tissue has been described. The derived pgMECs were maintained in cell culture for several passages without signs of further differentiation or senescence. The extensive characterization of established cell lines was performed and the main cell types in the mammary culture were determined. The pgMECs were capable of innate immune response and remained hormone responsive. Under lactogenic conditions, the cells successfully change morphology, synthesize milk proteins, and form lumen-like and milk drop–like structures. The established cell lines represent an adequate model of goat mammary tissue, useful for basic and applied

attributed to a better proliferation capability of such cells [38].

research in mammary gland biology and biotechnology.

**6. Conclusions**

(magnification 200×; scale bar = 50 μm).

Complex regulatory mechanisms are required for the onset of lactation, which involve functional differentiation/proliferation of the mammary cells and considerable anatomical and physiological tissue perturbations during gestation. Interestingly, milk proteins can be detected in pgMECs already after several days of growth in cell culture, even if derived from the tissue of nonlactating animals. It seems that pgMECs are capable of terminal differentiation from basal to secretory cells in a short period of time, when grown in environment enabling lactogenic differentiation. CSN2 was localized mainly in circular (lumen-like) structures, formed only by pgMECs grown in lactogenic conditions (**Figure 10B**). To conclude, expression of CSN2 in pgMECs is variable and depends mostly on starting tissue material and growth conditions.

#### *5.2.4. Lactating versus juvenile mammary tissue–derived pgMECs*

The expression of the steroid receptors and beta casein was the highest in juvenile mammary tissue–derived pgMECs, which could indicate a possible role of ER and PR in lactogenic

Development of an *In Vitro* Goat Mammary Gland Model: Establishment, Characterization, and… http://dx.doi.org/10.5772/intechopen.71853 181

**Figure 10.** Expression of CSN2 mRNA and localization of the protein in pgMECs (photo: J. Ogorevc). (A) RT-qPCR amplification plot for housekeeping GAPDH and CSN2 in three different pgMEC lines. (B) Beta casein was present in pgMEC lines and localized mainly in vacuoles (lumen-like structures), formed only under lactation-inducing conditions (magnification 200×; scale bar = 50 μm).

differentiation and proliferation of mammary epithelial cells. Research on humans shows that ER-/PR-positive mammary cells represent early mammary progenies and regulate differentiation of ER-negative mammary progenitors, from which basal/myoepithelial cells arose, and to ER-positive bipotent progenitors, which can give rise to luminal and myoepithelial lineage [33, 44]. It would make sense that progenitor fractions are enriched in juvenile mammary tissue (and in derived pgMECs) where lactogenic differentiation had not occurred yet. Prpar and colleagues [10] reported existence of different mammary progenitors in goats, luminalrestricted, myoepithelial-restricted, and bipotent and showed that the tissue from animals at the peak of lactation and from a juvenile animal contained the highest number of luminal progenitors, whereas the tissue at the onset of involution contained mostly myoepithelial progenitors. In our experience, tissue from young, nonlactating (juvenile) goats seems more suitable for preparation of lactation-competent cell cultures than tissue from lactating or involuting animals. Similar was also suggested in case of bovine primary mammary cells and attributed to a better proliferation capability of such cells [38].

#### **6. Conclusions**

*5.2.3. Expression of beta casein (CSN2)*

180 Goat Science

matrix for CSN2 expression [36].

and growth conditions.

*5.2.4. Lactating versus juvenile mammary tissue–derived pgMECs*

factors, and cell-cell and cell-substratum interactions.

Expression of caseins was detected in various primary and immortalized mammary cell lines from different species. For example, mouse HC11 cells [36], several bovine mammary cell lines [15, 28, 37–39], and goat primary mammary cells [8, 18, 40] are able to express caseins. Transcription of CSN2 was studied in mouse HC11 cells and the authors [41] found that its expression is induced synergistically by combination of lactogenic hormones, local growth

We evaluated how the starting tissue material, addition of hormones (insulin, hydrocortisone, and prolactin) to the growth medium, and growth on a commercially prepared extracellular basement membrane matrix affect relative expression of beta casein [42], determined using RT-qPCR (**Figure 10A**). The CSN2 transcripts were detected in all of the samples, including cells originating from nonlactating goat, grown in basal medium. However, the expression of CSN2 and response to different growth conditions were different in individual cell lines. Interestingly, we found that CSN2 expression was the highest in pgMECs derived from juvenile goats, grown in lactogenic medium. Addition of hormones in most cases induced expression of CSN2. The effect of extracellular membrane matrix–covered growth surface (Geltrex in our case) was variable. We found that extracellular membrane matrix growth surface was not indispensable for casein expression in the cell lines. In some of the cell lines, membrane matrix significantly increased CSN2 expression, whereas in others, it did not have a statistically significant effect (in several cases, expression of CSN2 was even lower [nonsignificantly] in pgMECs grown on membrane matrix). We speculate that some of the cell types present in a heterogeneous mixture of the cell cultures, are capable of extracellular matrix production in quantities sufficient for luminal cells to achieve lactation competency. It was reported previously that mouse HC11 cells can produce extracellular matrix [43] and do not require additional

Complex regulatory mechanisms are required for the onset of lactation, which involve functional differentiation/proliferation of the mammary cells and considerable anatomical and physiological tissue perturbations during gestation. Interestingly, milk proteins can be detected in pgMECs already after several days of growth in cell culture, even if derived from the tissue of nonlactating animals. It seems that pgMECs are capable of terminal differentiation from basal to secretory cells in a short period of time, when grown in environment enabling lactogenic differentiation. CSN2 was localized mainly in circular (lumen-like) structures, formed only by pgMECs grown in lactogenic conditions (**Figure 10B**). To conclude, expression of CSN2 in pgMECs is variable and depends mostly on starting tissue material

The expression of the steroid receptors and beta casein was the highest in juvenile mammary tissue–derived pgMECs, which could indicate a possible role of ER and PR in lactogenic The development of primary cell lines from lactating, juvenile, and involuting goat mammary tissue has been described. The derived pgMECs were maintained in cell culture for several passages without signs of further differentiation or senescence. The extensive characterization of established cell lines was performed and the main cell types in the mammary culture were determined. The pgMECs were capable of innate immune response and remained hormone responsive. Under lactogenic conditions, the cells successfully change morphology, synthesize milk proteins, and form lumen-like and milk drop–like structures. The established cell lines represent an adequate model of goat mammary tissue, useful for basic and applied research in mammary gland biology and biotechnology.
