SHOWCASE ON RESEARCH
Cells in Human Milk – What Do They Tell Us
Alecia-Jane Twigger* and Peter Hartmann
School of Chemistry and Biochemistry, University of Western Australia, Perth, WA 6009
*Corresponding author: [email protected]
cells that can be used to non-invasively characterise
lactating mammary cell gene expression, morphology
and regenerative capabilities (Fig. 1).
Early studies examining cells from human colostrum
(HC) and HM cells were conducted by smearing undiluted
samples across slides to morphologically determine cell
types and properties (7). Originally it was thought that
milk was dominated by leukocytes and ‘foam cells’
(colostrum bodies) (7), however more modern techniques
of cell isolation and classification have advanced our
understanding of HM cells. Studies from our laboratory
using freshly isolated cells extracted from either HC or
HM examined for CD45 with flow cytometry found that
whilst leukocytes dominate HC, they revert to <2.5% of
total mature HM cells when both the mother and infant
are healthy (8). On the other hand, infection of either
the infant or mother can affect the leukocyte content
of HM, increasing slightly for infant only infections
and more dramatically (>90% leukocyte content) in
cases of maternal mastitis (8,9) (Fig. 2). These findings,
together with animal studies illustrating immune cell
transfer through the milk to various infant organs (10),
particularly the thymus (11,12),
suggests a potential protective
and programming role of these
cells for the infant. Additionally,
the heightened levels of immune
cells in cases of maternal mastitis
provides a unique opportunity
to study pathways of mammary
gland defence that may be
optimised for rapid resolution of
mammary gland infection.
Unlike bovine milk, which
contains low numbers of
viable cells (primarily immune
cells) (13,14), mature HM is
a rich source of viable cells,
representative of the lactating
Stem cell
mammary epithelium (3,15)
(Fig. 3A). Numerous viable
cells are available for isolation
from mature HM, ranging
Common
from 1.16x104 – 3.9x106 cells per
progenitor
millilitre of milk, of which 76100% are viable (16-18). Lower
Luminal
numbers of viable cells can be
Myoepithelial
progenitor
progenitor
isolated from either HC or early
HM samples (18). Variation in
these values are likely due to
differences in isolation protocols,
where storage and centrifugation
speed
are
important
in
Myoepithelial
Alveolar
Ductal
The physiological processes of human lactation are
not as foolproof as is often thought, as many women
experience milk production difficulties, often leading to
early weaning. Most of these difficulties are attributed
to pain during feeding and real or perceived low
milk supply. Some identified risk factors for impaired
lactation include maternal obesity resulting in abnormal
development of the mammary gland (1) and maternal
mammary ZnT2 transporter mutations being linked to
low production (2). Evidently, greater research into the
mechanisms facilitating successful morphogenesis of
the gland during pregnancy and lactation is desperately
required to develop strategies to assist at-risk women
and optimise their milk production. In the past, studies
into the development of the gland have been hindered by
the lack of readily accessible human lactating tissue (3).
As a result, many previous studies have instead focused
on animal models of pregnancy and lactation, although
major differences exist in mammary gland development
(4), gene expression (5) and milk composition (6) between
mammalian species. However, human milk (HM)
contains immune, stem and differentiated mammary
Figure 1
Fig. 1. Cells isolated from
human milk contain a
hierarchy of stem and
differentiated mammary cells.
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SHOWCASE ON
RESEARCH
Cells in Human Milk
determining the number and viability of extracted cells
(19). Recently, polyploidal luminal cells have been
identified in the human mammary gland and these play a
vital role in lactation, as observed by low milk production
in mice with inhibited binucleated cell formation (20). It
appears polyploidal cells filter into the milk as seen in
Fig. 3A (Twigger, unpublished data), although more
detailed imaging should be carried out to confirm this.
Examination of HM cells has confirmed the expression of
many key mammary markers including integrin alpha-6
(CD49f), membrane metalloendopeptidase (CD10),
epithelial adhesion marker (EPCAM) and cytokeratins
5, 14, 18 and 19 (CK5, CK14, CK18 and CK19) which are
representative of mammary stem and differentiated basal
and luminal cells (16,21,22). Furthermore, a previous
study found that mammary tissue and milk cells from
lactating macaques had comparable gene expression,
suggesting milk cells to be representative of mammary
tissue (23). Discrepancies in HM cell gene expression
have been associated with different characteristics of the
mother and infant, building our understanding of the
influencing factors that determine successful mammary
gland development and milk production.
The population of cells in HM vary in composition over
the course of a breastfeed as well as within and between
women, much like milk composition in general (3). HM
cell content varies over the course of a breastfeed, similar to
the variation observed in fat content in HM, being greater
towards the end of a feed and peaking approximately 30
minutes post-feeding (17). In a recent study, variation
of HM cell gene expression was established between 66
participants for 17 different genes representative of the
mammary cellular hierarchy (16). Gene expression was
investigated in light of maternal and infant demographics,
which included maternal body mass index (BMI), breast
volume, gestational age of the infant and lactation stage,
in order to determine the presence of any associations
(16). Of note, lower expression of CK18, which is
representative of lactocytes, was associated with higher
maternal BMI, potentially suggesting undifferentiated
lactocytes as an underlying cause of low milk production
in obese mothers (16). In addition, higher expression of
estrogen related receptor beta (ESRRB) expression and
lower expression of growth differentiation factor 3 (GDF3)
expression were associated with earlier stages of lactation,
implying ongoing development of the mammary gland
over the course of lactation. Indeed whole transcriptome
analysis between milk fat globules isolated from HC,
transitional HM and mature HM found differences in
expression of key genes such as milk protein genes alphalactatalbumin and beta casein between these three stages
of lactation (24). Moreover, immune defence pathways
were expressed specifically in HC, upregulation of milk
synthesis machinery was found in transitional HM and
lipid production pathways were dominant in mature HM
(24). A subsequent study examined changes in the HM cell
transcriptome by comparing sequential samples collected
over the first 12 months of lactation with prepartum
secretions from the same women and resting mammary
gland tissue (25). As well as confirming findings from the
HM fat globule study, this study determined differences
in the expression of cell adhesion pathway genes between
prepartum secretion cells and resting tissue, as well as
differences in the expression of cancer pathway genes
between mature HM cells and resting mammary tissue
that were not identified in the HM fat globule study
(25). Studies such as these will elucidate the mechanisms
behind human milk synthesis and mammary metabolism
that are trigged during secretory activation. It is evident
that not only can HM cells be utilised to examine
mammary gene expression (16,26,27), they can also be
grown in culture to help further profile the developing
and functioning mammary gland (21,22,26,28,29).
The first studies of HM cell culture were conducted
in the late 1970s and early 1980s, where cells isolated
from early milk (3 to 7 days post-partum) proliferated
in 2D culture and survived multiple passages (30-33).
Interestingly, it was noted that not all cells participated
in cell division whilst many of the cells could proliferate
for 8–11 passages (30). Subsequent studies found cells
isolated from more mature HM could also be cultured
and these displayed many of the features originally
B i)
B ii)
Count
Side Scatter
A
i
iii
70.5% CD45+
55%
CD45+
ii
Forward Scatter
CD45
CD45
Fig. 2. Flow cytometric analysis of isolated milk cells collected from a woman with mastitis.
A. Forward and side scatter plots reveal size and granularity of milk cells which were gated to examine
subpopulations i), ii) and iii).
B. Cells from subpopulations i) and ii) were examined for CD45, where subpopulation i) had 70.5% of cells
positive for CD45, whereas subpopulation ii) had 55% of cells positive for CD45.
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SHOWCASE ON
RESEARCH
Cells in Human Milk
Fig. 2. Human milk cells examined with an inverted microscope.
A. Freshly isolated cells stained with Trypan Blue exclusion dye illustrate a large number of viable cells with some
apparent bi-nucleated cells (red arrows), non-viable cells (black arrow) and few milk fat globules (yellow arrow).
B. Cells placed in mammary media for two days begin to forming spheroids under non-adherent conditions.
described with added immunofluorescence staining,
demonstrating the diversity in the resulting mammary
cell subtypes (21,22,26,28). Additionally, it was found
that CK5 and CK14 gene expression was enriched after
culture, providing an opportunity to select and expand
the progenitor cell population from freshly isolated HM
cells (21). Another study using cultured monolayer HM
cells was able to identify differences in the morphology
and growth patterns of the cells after transfection of 14-33s (Sigma) siRNA (28). Similar gene silencing techniques
could be utilised in the future to examine the importance
of specific genes for mammary gland development.
Using non-adherent culturing conditions, 3D structures
can also be formed from HM cells (Fig. 3B) to create bilayered spheroids containing CD49f+ positive basal cells
and Prl-R+ (prolactin receptor) luminal cells, mimicking
mammary alveoli (22). These 3D structures were shown
to synthesise and secrete the milk proteins alphalactalbumin and lactoferrin into the culture media (26).
Enhancements of these protocols may utilise the latest
organoid culture methods, which are able to produce
organised tertiary alveolar and conn3cting ductal
structures from resting mammary cells (34). Indeed, HM
cell culture may hold the key to a greater understanding
of human mammary gland development, which is driven
by progenitor and stem cells.
Currently, human mammary stem cells are not well
defined with respect to markers, location or differentiation
capabilities due to the limited availability of tissue and
heterogeneous methods of investigation (35). It is likely
that a spectrum of mammary stem cells exists with
different differentiation capabilities. Investigations of
lactating mammary cells isolated through HM have
found subpopulations that are positive for markers
associated with bipotent, multipotent and pluripotent
markers (21,26,28). Nestin was the first multipotent stem
cell marker identified in HM cells and co-stained with the
mammary stem cell marker CK5 (21). HM cells positive
for multipotent marker p63 have also been identified and
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can differentiate into two separate mammary cell lineages
and consequently undergo cell cycle arrest (28). Lineagespecific progenitor markers such as CD105, CD29, CD90
and Stro-1, which are representative of mesenchymal stem
cells, have been identified in HM, which are able to secrete
vascular endothelial growth factor (VEGF) in culture (3639). Whether these cells are true mesenchymal stem cells is
debatable (40), however further studies suggest that HM
cells exhibit lineage plasticity. An interesting study by
Hassiotou et al. demonstrated the presence of pluripotent
transcription factors OCT4, SOX2 and NANOG in HM
cells and illustrated their plasticity by differentiating
them into neuronal, hepatocyte, pancreatic and bone–like
cells (26). Similar studies have been conducted in resting
mammary tissue, which also contain highly plastic cells
(41), albeit at lower densities (26). Evidently, stem cells
in the mammary gland are vital to achieving maturity in
pregnancy and to some degree lactation. However it is
not known why these cells are excreted into the milk. It is
possible that they exist for the benefit of the infant, where
they have been found to survive the gastrointestinal tract
of the offspring and are transferred to distant tissues
where they integrate to potentially contribute to tissue
function (42). It has been speculated that HM cells have
a potential use in regenerative therapies; however a
great deal more work is needed to understand the cells’
characteristics and proportions in milk.
Clearly HM contains a fascinating cellular composition
that can be non-invasively isolated and used in a plethora
of experiments to examine lactating mammary cell gene
expression, growth and in vitro morphology. HM cells
will also assist in the understanding of the intricacies of
mammary gland development and function. Future milk
cell work investigating the characteristics of normal HM
cells hold the promise of unravelling mammary gland
dysfunction and pathologies such as low milk production
and mastitis, both of which are major contributors to
early weaning.
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SHOWCASE ON
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Cells in Human Milk
References
1. Rasmussen, K.M. (2007) Ann. Rev. Nutr. 27, 103-121
2. Dempsey, C., McCormick, N.H., Croxford, T.P., Seo,
Y.A., Grider, A., and Kelleher, S.L. (2012) J. Nutr. 142,
655-660
3. Hassiotou, F., Geddes, D.T., and Hartmann, P.E. (2013)
J. Hum. Lact. 29, 171-182
4. Dontu, G., and Ince, T.A. (2015) J. Mammary Gland Biol.
Neoplasia 20, 51-62
5. Lemay, D.G., Lynn, D.J., Martin, W.F., Neville, M.C.,
Casey, T.M., Rincon, G., Kriventseva, E.V., Barris, W.C.,
Hinrichs, A.S., Molenaar, A.J., Pollard, K.S., Maqbool,
N.J., Singh, K., Murney, R., Zdobnov, E.M., Tellam,
R.L., Medrano, J.F., German, J.B., and Rijnkels, M. (2009)
Genome Biol. 10, R43
6. Malacarne, M., Martuzzi, F., Summer, A., and Mariani,
P. (2002) Int. Dairy J. 12, 869-877
7. Holmquist, D.G., and Papanicolaou, G.N. (1956) Ann.
NY Acad. Sci. 63, 1422-1435
8. Hassiotou, F., Hepworth, A.R., Metzger, P., Lai, C.T.,
Trengove, N., and Hartmann, P.E. (2013) Clin. Transl.
Immunology 2, e3
9. Riskin, A., Almog, M., Peri, R., Halasz, K., Srugo, I., and
Kessel, A. (2012) Pediatr. Res. 71, 220-225
10.Jain, L., Vidyasagar, D., Xanthou, M., Ghai, V., Shimada,
S., and Blend, M. (1989) Arch. Dis. Child 64, 930-933
11.Zhou, L., Yoshimura, Y., Huang, Y., Suzuki, R.,
Yokoyama, M., Okabe, M., and Shimamura, M. (2000)
Immunology 101, 570-581
12.Ghosh, M.K., Nguyen, V., Muller, H.K., and Walker,
A.M. (2016) J. Immunol. 197, 2290-2296
13.Lee, C.-S., Wooding, P., and Kemp, P. (1980) J. Dairy
Res. 47, 39-50
14.Boutinaud, M., and Jammes, H. (2002) Reprod. Nutr.
Dev. 42, 133-147
15.Ho, F.C., Wong, R.L., and Lawton, J.W. (1979) Acta
Paediatr. Scand. 68, 389-396
16.Twigger, A.J., Hepworth, A.R., Lai, C.T., Chetwynd, E.,
Stuebe, A.M., Blancafort, P., Hartmann, P.E., Geddes,
D.T., and Kakulas, F. (2015) Sci. Rep. 5, 12933
17.Hassiotou, F., Hepworth, A.R., Williams, T.M., Twigger,
A.J., Perrella, S., Lai, C.T., Filgueira, L., Geddes, D.T.,
and Hartmann, P.E. (2013) PLoS One 8, e78232
18.Gaffney, E.V., Polanowski, F.P., Blackburn, S.E., and
Lambiase, J.P. (1976) Cell Tissue Res. 172, 269-279
19.Hassiotou, F., Savigni, D.L., Hartmann, P., and Geddes,
D.T. (2015) FASEB J. 29
20.Rios, A.C., Fu, N.Y., Jamieson, P.R., Pal, B., Whitehead,
L., Nicholas, K.R., Lindeman, G.J., and Visvader, J.E.
(2016) Nat. Commun. 7, 11400
21.Cregan, M.D., Fan, Y., Appelbee, A., Brown, M.L.,
Klopcic, B., Koppen, J., Mitoulas, L.R., Piper, K.M.,
Choolani, M.A., Chong, Y.S., and Hartmann, P.E. (2007)
Cell Tissue Res. 329, 129-136
Page 12
22.Thomas, E., Lee-Pullen, T., Rigby, P., Hartmann, P., Xu,
J., and Zeps, N. (2012) Stem Cells 30, 1255-1264
23.Lemay, D.G., Hovey, R.C., Hartono, S.R., Hinde, K.,
Smilowitz, J.T., Ventimiglia, F., Schmidt, K.A., Lee, J.,
Islas-Trejo, P., Silva, P.I., Korf, I., Medrano, J., Barry,
P.A., and German, J.B. (2013) BMC Genomics 14, 872
24.Lemay, D.G., Ballard, O.A., Hughes, M.A., Morrow,
A.L., Horseman, N.D., and Nommsen-Rivers, L.A.
(2013) PLoS One 8, e67531
25.Twigger, A.J., Geddes, D.T., and Kakulas, F. (2016) 18th
International Society for Research into Human Milk
and Lactation Conference, Breastfeeding Medicine,
Stellenbosch
26.Hassiotou, F., Beltran, A., Chetwynd, E., Stuebe, A.M.,
Twigger, A.J., Metzger, P., Trengove, N., Lai, C.T.,
Filgueira, L., Blancafort, P., and Hartmann, P.E. (2012)
Stem Cells 30, 2164-2174
27.Sharp, J.A., Lefevre, C., Watt, A., and Nicholas, K.R.
(2016) Funct. Integr. Genomics 16, 297-321
28.Thomas, E., Zeps, N., Cregan, M., Hartmann, P., and
Martin, T. (2011) Cell Cycle 10, 1-7
29.Hosseini, S.M., Talaei-Khozani, T., Sani, M., and
Owrangi, B. (2014) Neurol. Res. Int. 2014, 807896
30.Chang, S.E., Keen, J., Lane, E.B., and TaylorPapadimitriou, J. (1982) Cancer Res. 42, 2040-2053
31.Stoker, M., Perryman, M., and Eeles, R. (1982) Proc. R.
Soc. Lond. B. Biol. Sci. 215, 231-240
32.Gaffney, E.V. (1982) Cell Tissue Res. 227, 563-568
33.Taylor-Papadimitriou, J., Shearer, M., and Tilly, R.
(1977) J. Natl. Cancer Inst. 58, 1563
34.Linnemann, J.R., Miura, H., Meixner, L.K., Irmler, M.,
Kloos, U.J., Hirschi, B., Bartsch, H.S., Sass, S., Beckers, J.,
Theis, F.J., Gabka, C., Sotlar, K., and Scheel, C.H. (2015)
Development 142, 3239-3251
35.Visvader, J.E., and Stingl, J. (2014) Genes Dev. 28, 11431158
36.Fan, Y., Chong, Y.S., Choolani, M.A., Cregan, M.D., and
Chan, J.K. (2010) PLoS One 5, e14421
37.Indumathi, S., Dhanasekaran, M., Rajkumar, J. S., and
Sudarsanam, D. (2012) Cytotechnology 65, 385-393
38.Patki, S., Kadam, S., Chandra, V., and Bhonde, R. (2010)
Hum. Cell 23, 35-40
39.Kaingade, P.M., Somasundaram, I., Nikam, A.B., Sarang,
S.A., and Patel, J.S. (2016) Breastfeed. Med. 11, 26-31
40.Kakulas, F., Geddes, D. T., and Hartmann, P. E. (2016)
Breastfeed. Med. 11, 150-151
41.Roy, S., Gascard, P., Dumont, N., Zhao, J., Pan, D.,
Petrie, S., Margeta, M., and Tisty, T. (2013) Proc. Natl.
Acad. Sci. USA 110, 4598-4603
42.Hassiotou, F., Mobley, A., Ocal, O., Filgueira, L.,
Geddes, D.T., Hartmann, P.E., and Wilkie, T.M. (2014)
17th Conference of International Society for Research in
Human Milk and Lactation
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