Cellular Components,Including Stem-Like Cells,of Preterm Mother's Mature Milk as Compared with Those in Her Colostrum: A Pilot Study

Abstract

Purpose and Study Objective:

Whether the preterm mothers' mature milk retains the same cellular components as those in colostrum including stem-like cell, cell adhesion molecules, and immune cells.

Participants:

A total of five preterm mothers were recruited for the study having an average age of 30.2 years and gestational age of 29.8 weeks from the Pristine Women's Hospital, Kolhapur. Colostrum milk was collected within 2–5 days and matured milk was collected 20–30 days after delivery from the same mothers.

Methodology:

Integral cellular components of 22 markers including stem cells, immune cells, and cell adhesion molecules were measured using flowcytometry.

Outcome:

Preterm mature milk was found to possess higher expressions of hematopoietic stem cells, mesenchymal stem-like cells, immune cells, few cell adhesion molecules, and side population cells than colostrum.

Conclusion:

The increased level of these different cell components in mature milk may be important in the long-term preterm baby's health growth. Further similar research in a larger population of various gestational ages and lactation stages of preterm mothers is warranted to support these pilot findings.

Introduction

Human breast milk is a dynamic fluid composed of macro- and micronutrients and other bioactive factors specially suited to meet the needs of the newborn for its growth and development.^1,2 Besides these components, breast milk possesses several cellular components including colostral corpuscles, polymorphonuclear leukocytes, mononuclear phagocytes, lymphocytes, and the stem cells.^3–5 These components of breast milk form complex mixtures that contribute to the beneficial effects of breastfeeding. The particular benefits for preterm infants include protection from neonatal sepsis and necrotizing enterocolitis, lower rates of retinopathy in prematurity, and improved developmental outcomes.^6,7 Breastfeeding particularly of colostrum is, therefore, beneficial for the health of preterm infants.^8,9 This pilot study aimed to compare the quantity of various integral cellular components (hematopoietic, mesenchymal stem-like cells, cell adhesion molecules, epithelial cells, immune cells, and so on) of mature milk with those of colostrum, so as to examine whether these cell components are retained over time in mature milk.

Materials and Methods

Colostrum and mature milk were collected from five preterm mothers. The preterm delivered mothers with an average age of 30.2 years and gestational age 29.8 weeks were recruited from the Pristine Women's Hospital, Kolhapur, Maharashtra, India, for the study. Clinical correlations pertaining to this study population are mentioned (Table 1).

Table 1.

Clinical Data of the Study Population

S. No	Age of mother	Gestational age (weeks)	Birth weight of infant (g)	APGAR score at 1 minute	Day and mL of colostrum collected	Day and mL of mature milk collected
1	28	29	1.628	7.8	2, 6.5	25, 8
2	32	29	1.902	7.1	3, 7.0	28, 8.5
3	30	29	2.029	7.9	3, 6.0	30, 7.2
4	33	30	1.898	7.2	5, 5.5	22, 7.0
5	28	32	1.600	7.4	3, 7.0	29, 6.5

Preterm colostrum and mature milk samples (n = 5) were collected manually in an aseptic manner. The preterm colostrum was collected within 2–5 days of delivery and the mature milk sample was collected within 20–30 days after delivery from the same mothers. All samples were processed within 2 hours of collection. This study was approved by the Institutional Ethics Committee, Pristine Women's Hospital. The purpose of this research was explained to all mothers under study and formal written consent was obtained before collection.

Processing of human breast milk

All the samples were processed similar to our previously published article.⁵ The samples were diluted with Dulbecco's Modified Eagle's Medium low glucose and centrifuged at 400 g for 10 minutes. The cellular component obtained in the form of a pellet was collected and washed twice with phosphate buffer saline (PBS). The final cellular constituent pelleted was subjected to analysis of cell surface antigenic expression profile using flowcytometry.

Flowcytometry characterization

Flowcytometry was performed on a Becton Dickinson fluorescence-activated cell sorter Aria (www.bd.com) using a 488-nm argon ion laser and 632-nm red laser for excitation. Fluorescence emission was collected using the corresponding detectors. About 1 × 10⁶ cells of colostrum and breast milk were stained with saturating concentrations of fluorochrome-conjugated antibodies (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/bfm). The cells were incubated in the dark for 20 minutes at room temperature, washed thrice with wash flow buffer, and resuspended in 500 μL of PBS. Data acquisition and analysis were then performed using DIVA Software (Becton Dickinson). A minimum of 10,000 events were characterized and recorded.

Statistical analysis

Software-assisted (statistical package for social science SPSS 20.0-SPSS, Inc., Chicago, IL) analysis of data by paired Student's one tailed test was carried out between the results of preterm colostrum and breast milk samples. The results are expressed as mean ± standard error of mean (SEM). In all comparisons, p values **<0.01 and *<0.05 were considered to be significant.

Results

Flowcytometric analysis

Cellular constituents in preterm colostrum and breast milk

Preterm colostrum (n = 5) and mature milk samples (n = 5) possess heterogeneous cellular constituents of both stem-like cells and nonstem cell populations characterized by cell surface marker profiles: CD34, CD133, CD117 (hematopoietic stem cells); CD90, CD105, CD73 (mesenchymal stem cells); CD29, CD44 (myoepithelial cells); CD209, CD86, CD83, CD14, CD13, HLADR, CD45 (immune cells); as well as cell adhesion molecules (CD31, CD54, CD166, CD106, CD49d); and other markers (CD140b, ABCG2) identified using flowcytometry (Supplementary Figs. S1 and S2). The expression profiles of these markers are represented as mean ± SEM with p values *<0.05 and **<0.01 taken to be of statistical significance (Table 2).

Table 2.

Expression Profile of Preterm Colostrum and Mature Milk

		Mean ± SEM
Properties	Antigen	Preterm colostrum	Preterm mature milk	p
Hematopoietic stem/progenitor cells	CD34^**	11.5 ± 1.2	40.7 ± 1.877	0.0006
	CD133	33.7.4 ± 3.93	26.96 ± 1.65	0.02
	CD117^**	12.36 ± 2.13	25.12 ± 1.74	0.0003
Mesenchymal stem cells	CD90^**	7.10 ± 1.02	25.72 ± 2.81	0.0008
	CD105^**	23.16 ± 4.49	53.06 ± 3.10	0.0005
	CD73	2.1 ± 0.15	2.36 ± 0.11	0.114
Myoepithelial cells	CD29^*	63.13 ± 4.32	70.68 ± 3.2	0.03
	CD44^**	30.14 ± 2.31	19 ± 1.23	0.0004
Monocytes/macrophages/dendritic cells	CD86^**	25.89 ± 2.92	11.65 ± 1.88	0.0007
	CD209^**	2 ± 0.23	14.38 ± 1.44	0.0001
	CD83^**	11.08 ± 2.83	17.28 ± 1.59	0.002
	CD14^**	48.4 ± 3.17	90.5 ± 2.94	0.0008
	CD13^**	30.9 ± 2.04	50.78 ± 3.13	0.0002
	HLADR^**	31.11 ± 2.75	59 ± 2.91	0.0001
	CD45	55.31 ± 5.24	11.69 ± 0.87	0.00002
Cell adhesion molecules	CD31	18.8 ± 2.56	9.45 ± 1.41	0.0002
	CD54^**	42.36 ± 2.54	50.56 ± 2.34	0.004
	CD166^**	9.1 ± 0.86	16.4 ± 1.85	0.0004
	CD106^*	3.14 ± 0.23	11.32 ± 1.68	0.0001
	CD49d	13.88 ± 3.03	16.74 ± 3.04	0.10
Side population	ABCG2^**	2.14 ± 0.11	21.77 ± 4.08	0.0002
Growth factor	CD140b^**	40.14 ± 2.42	50.62 ± 0.62	0.0001

Values are represented as mean ± SEM.

p < 0.05 and ^**p < 0.01 for increased level in mature milk are highlighted in bold.

SEM, standard error of mean.

Expression of stem cell population

Our results showed less expression of CD90 in preterm colostrum (7.10 ± 1.02) than in mature milk (25.72 ± 2.81). There was also a higher expression of CD105 in preterm mature milk (53.06 ± 3.10) than in colostrum (23.16 ± 4.49) and a sparse expression of CD73 in both colostrum (2.1 ± 0.15) and mature milk (2.36 ± 0.11). In the case of hematopoietic stem/progenitor cells, the expression of CD34 and CD117 in preterm mature milk (40.7 ± 1.877 and 25.12 ± 1.74, respectively) was higher than that in colostrum (11.5 ± 1.2 and 12.36 ± 2.13, respectively). There was moderate expression of CD133 in both preterm colostrum (33.7.4 ± 3.93) and mature milk (26.96 ± 1.65). The difference between levels of expression in preterm mature milk and colostrum was statistically significant (Table 2).

Expression of cell adhesion molecules

The samples showed higher expression of CD31 in preterm colostrum (18.8 ± 2.56) than in mature milk (9.45 ± 1.41). The expression of CD106 was sparse in preterm colostrum (3.14 ± 0.23) than in mature milk (11.32 ± 1.68). Low expression of CD49d was found in both colostrum (13.88 ± 3.03) and mature milk (16.74 ± 3.04). The expression of CD166 was low in mature milk (16.4 ± 1.85) and sparse in colostrum (9.1 ± 0.86). Of all cell adhesion molecules, CD54 was highly expressed in both mature milk (50.56 ± 2.34) and colostrum (42.36 ± 2.54) with a significant p value of <0.05 (Table 2).

Expression of immune cells

This study revealed moderate expression of CD86 in preterm colostrum (25.89 ± 2.92) and low expression in mature milk (11.65 ± 1.88). Similarly, a low expression of CD209 and CD83 was observed in preterm mature milk (14.38 ± 1.44 and 17.28 ± 1.59, respectively), whereas colostrum revealed sparse expression (2 ± 0.23 and 11.08 ± 2.83, respectively). Remarkable expression of CD14 was observed in mature milk (90.5 ± 2.94) with a moderate expression in colostrum (48.4 ± 3.17). CD13 and HLA-DR were highly expressed in preterm mature milk (50.78 ± 3.13 and 59 ± 2.91, respectively) than in colostrum (30.9 ± 2.04 and 31.11 ± 2.75, respectively). The expression of CD45 was high in colostrum (55.31 ± 5.24) but was found to be low in mature milk (11.69 ± 0.87) (Table 2).

Myoepithelial/integrins and other unique markers

The study recorded the expression of integrin CD29, which was high in both colostrum and mature milk (63.13 ± 4.32 and 70.68 ± 3.2, respectively). There was moderate expression of CD44 in colostrum (30.14 ± 2.31) with a low expression in mature milk (19 ± 1.23). The expression of CD140b, the platelet-derived growth factor receptor b, was high in breast milk (50.62 ± 0.62) and expressed moderately in colostrum (40.14 ± 2.42). In addition, ABCG2, the side population marker, was also high in mature milk (21.77 ± 4.08) than in colostrum (2.14 ± 0.11) (Table 2).

Discussion

The increase in cell components identified in mature milk over colostrum include stem-like cells, epithelial cell, some cell adhesion molecules, and other cell population, probably favoring immunity, growth, and development of premature infants. In brief, the immune markers were higher in mature milk than in colostrum, except for the marker CD45. Other immune markers such as CD209 and CD83 were present in the samples of colostrum but not in mature milk. This suggests that leucocytes and other dendritic/costimulatory markers play a vital role in immunity in the early days of colostral feeding as compared with mature milk, whereas the monocytes/major histocompatibility complexes specifically might render immunity later. Further studies on the existence of this different immune cell population between colostrum and mature milk may throw light on understanding the development of premature babies and their immune capacity in fighting infections.

Our results demonstrated a higher expression of hematopoietic stem-like cells in mature breast milk than in colostrum. However, the expression pattern of CD133, hematopoietic progenitor cell, was similar in both colostrum and breast milk. Although preterm colostrum expressed a sparse expression of mesenchymal stem-like cells, CD90 and CD105 were shown to be expressed slightly higher in mature milk. It is presumed that upon culture, these cells would become true mesenchymal stem cells through epithelial mesenchymal transition as reported.¹⁰ The higher expressions of CD90 and CD105 in mature milk could be correlated with other functions such as immunity, immunomodulation, host-defense mechanism, anti-inflammatory actions, angiogenic activity required for normal growth, and development coupled with some endothelial functions.^11,12 The preterm colostrum results obtained from our study with regard to hematopoietic stem cells and mesenchymal stem-like cells were higher than that reported in the literature.^4,5,13

With regard to cell adhesion molecule, CD54 shows the highest expression than other population. Although it could be presumed that CD105 and CD31 might promote transendothelial migration and vascular permeability and possible angiogenic activity for growth and development, further extensive research on the presence of an array of various adhesion molecules in different proportions toward their role in homing ability is warranted.

It is predicted that high expression of CD44 is required for firm adhesion of cell to endothelium. Data show that CD44 is activated by platelet-derived growth factor,¹⁴ which plays an important role in exogenous migration to injured site by interaction with hyluronate. Besides, CD13 was also identified to be a potent marker that plays a vital role in angiogenesis and migration apart from its immune activity.¹⁵ These features are of significance in development of premature babies and their blood circulation, making the need for long-term breastfeeding of preterm infants.

Limitations

This is a pilot study involving a small population.

Conclusion

This study identifies the differences in the expression pattern of various cellular components between preterm colostrum and mature milk. Both preterm colostrum and mature milk are enriched with integral cellular components; however, mature milk possesses higher expression of most integral cellular components as reported. These preliminary results warrant further research in infants of various gestational ages and lactating stages of women.

Footnotes

Acknowledgments

We thank all the lactating women participants for kindly providing colostrum and breast milk samples. We also thank Dr. Sachin Kulkarni, Pristine Women's Hospitals.

Disclosure Statement

No competing financial interests exist.

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