They will be famous: Multipotent stem cells in breast milk

Abstract

Breast milk represents the gold standard for neonatal nutrition, especially for preterm and term infants with a low birthweight. This awareness is based not only on the nutritional properties of human milk, which is specifically designed for the growth of humans but also on breast milk’s non-nutritional properties, such as protection against infection. In fact, breast milk should be considered a heterogeneous ecosystem, including a wide range of cells in addition to those involved in immune function; growth factors, such as vascular endothelial growth factor; multiple noncoding microRNAs; immune cells; epithelial cells and multipotent mesenchymal stem cells. This recent identification of a pool of progenitor stem cells in human milk is the driving force behind the growing research aimed at identifying the nature of these stem/progenitor cells and their sources.

Key Words: Stem cells; Breast milk; Neonate; Multipotent; Nutrition

Core Tip: The presence in the breast milk of a huge number of stem/progenitor cells, able to differentiate into so many different cell types, and their availability at a very low cost without the necessity of obtain complex permissions for their use, has induced in recent years multiple authors to consider the use of maternal milk stem cells in clinical practice. The possible fields of application of milk stem cells are so numerous, including all fields of regenerative medicine.

INTRODUCTION

Breast milk has long been considered an unrivalled source of nutrients, indispensable for the optimal growth of neonates[1]. Following this assumption, formulas have been considered for many years as possible substitutes for human milk. However, neonatologists around the world have cited breast feeding as the best choice for perinatal development of newborns, particularly for preterm and term infants with a low birthweight[2]. A recent meta-analysis on the advantages of breastfeeding for both mothers and infants confirmed the ability of lactation to protect against child infections and malocclusion. Moreover, interesting data emerged from a recent study on the anti-inflammatory and antiallergic properties of colostrum[3]. This study shows how innate and adaptive immune deficiency, with reduced immunological competence particularly evident in preterm infants, can be counteracted by colostrum.

Furthermore, advantages later in life have been confirmed by multiple studies of breastfed infants, including enhanced intellectual skills and reductions in overweight and diabetes in adulthood[4]. In the same meta-analysis, breastfeeding was reported to protect mothers against type 2 diabetes and ovarian cancer insurgence later in life. Approximately 17 years ago, some authors have started to consider this view of maternal milk restricted to its peculiar composition of nutrients as an old view, given the discovery of many types of cells, including multipotent mesenchymal cells, in milk[5]. The number of cells present in human milk was well evidenced by further studies, revealing that human colostrum contains approximately 5 million cells per ml. As a consequence, a breast-fed newborn may ingest approximately 10⁸ milk cells per day[6]. The discovery of a considerable number of cells in maternal human milk highlighted the necessity for a revised perspective on the nature of human milk, which, together with its distinctive nutritional composition, including proteins, lipids, carbohydrates, vitamins, and minerals, should also be regarded as a heterogeneous ecosystem, encompassing a diverse range of cells beyond those involved in immune function[6]. Here, we summarize the most relevant findings on maternal milk stem/progenitor cells discovered in recent years.

THE NATURE OF MATERNAL MILK CELLS

Cytological analysis of human milk composition in the first postpartum week revealed that breast milk is a more dynamic physiological fluid with a more complex composition than previously thought. The well-known necessary nutrients are associated with growth factors, such as vascular endothelial growth factor, multiple noncoding microRNAs, immune cells, epithelial cells and multipotent mesenchymal stem cells[7,8].

The identification of a stem progenitor cell pool in human milk promoted studies aimed at identifying the nature of these stem/progenitor cells and their source[9]. As shown in Figure 1, the maternal milk stem/progenitor pool emerged as a more complex pool than previously thought, including mesenchymal stem cells and embryonic stem cells. As shown in Figure 2, the heterogeneity of milk stem cells was first demonstrated by an immunohistochemical study aimed at identifying markers of human milk progenitor cells[10,11]. In this study, the following pools of milk cells were identified: (1) Mesenchymal stem cells characterized by immunoreactivity for CD44, CD29, stem cell antigen-1, nestin and vimentin; (2) Luminal progenitor cells characterized by CD61 expression; (3) Basal progenitor cells expressing octamer-binding transcription factor 4, SRY-box transcription factor 2, and Nanog homebox; and (4) Stem cells characterized by the expression of CD133, a typical stem/progenitor marker.

Figure 1 Different example of cells isolated with from fresh human milk. A-C: These cells, fixed and stained using our cytological methods routinely used in our laboratories, are characterized by different morphological aspects, underling the presence of a heterogeneous cell population. Briefly, breast milk samples were recruited from six healthy volunteers. The experimental analysis were authorized by the Comitato Etico Indipendente Ospedaliero (Università di Cagliari; Prot. PG/2022/795). Breast milk cells were isolated from fresh milk, diluted with an equal volume of Dulbecco’s phosphate-buffered saline (pH 7.4; Gibco) and centrifugated at 810 g for 20 minutes at 20 °C. The fat layer and liquid were removed, while the cell pellet was washed twice with Dulbecco’s phosphate-buffered saline. The pellet was fixed with ThinPrep solution (Hologic, Inc. www.hologic.com) and the cell samples were processed for immunocytochemistry analysis with a ThinPrep 5000 Processor. Different arrows highlight possible different cell types. Solid arrow: Stem cells; Hollow arrow: Immune cells; Triangle: Milk-secretory cells.

Figure 2 Immunocytochemistry staining. Different arrows highlight possible different cell types. Different arrows highlight possible different cell types. Antibodies for CD44 (SP37, Ventana 790-4537), CD133 (polyclonal, biorbyt, orb99113, 1:100), CD45 (LCA, 2B11 and PD7/26, Ventana 760-4279), Ki-67 (30-9, Ventana 790-4286), Wt1 (6F-H2, Cell Marque 760-4397), nestin (10C2, Sigma-Aldrich), Nanog (polyclonal, Abcam, ab80892, 1:100), OCT4 (MRQ-10, Cell Marque 760-4621), SOX2 (SP76, Cell Marque 760-4621), CK5 (SP27, Cell Marque 760-4935), CD34 (QBEnd/10, Ventana 790-2927), P63 (4A4, Ventana 790-4509), Thymosin β4 (Tβ4; polyclonal, abcam, ab14334, 1: 1000) and CK14 (SP23, Cell Marque 760-4805) were used for immunocytochemistry. Sample preparation was performed using standard validated cytologic protocols (ThinPrep Preserv Cyt solution, ThinPrep 5000 system, https://www.hologic.com). The Ventana automated stains system (http://diagnostics.roche.com) was used for immunocytochemistry staining and a qualitative analysis was performed by three different pathologists comparing the reactivity of different markers in slides of the same sample using an individual scoring system (morphological parameters, % of number of stained cells, area, and intensity of the stain). A: Immunocytochemistry staining for CD45; B: Immunocytochemistry staining for CD133; C: Immunocytochemistry staining for CD44. Solid arrow: Negative stem cell; Hollow arrow and triangle: Positive stem cell.

Interestingly, in these studies, the stem cell markers octamer-binding transcription factor 4, SRY-box transcription factor 2, and Nanog homebox were identified exclusively in lactating breast cells and were not expressed in the resting breast. This finding suggests the restriction of the activation of the breast stem cell pool during pregnancy, resulting in the production and secretion of pregnancy-specific stem cell progenitors into milk. More recent studies characterizing maternal milk stem cells revealed marked quantitative and qualitative differences between different lactation stages[12]. In that study, the percentages of haemopoietic stem/progenitor cells were significantly greater in mature milk than in colostrum and were influenced by gestational age at delivery. Another study suggested that ISL1 (a pioneer transcription factor that plays important roles in cell lineage specification and differentiation) could distinguish readily available sources of putative stem cells in human breast milk (Figure 3)[13].

Figure 3 A and B: ISL1 immunocytochemistry staining of some fresh human breast milk cells isolated using a ThinPrep^™ CytoLyt^™ solution and stained as described previously. Solid arrow: Positive stem cells; Hollow arrow and triangle: Negative stem cell.

These findings highlight the complexity of the dynamic nature of human milk stem cells[14]. At this juncture in the investigation of milk stem cells, several open questions have emerged: (1) Is there a hierarchy among milk stem/progenitors, and what is the role of milk stem cells in newborn survival and development? (2) Can maternal stem/progenitors migrate and integrate into neonatal organs, resulting in microchimaerism in every breastfed neonate? and (3) Can stem cells with maternal DNA differentiate into different cell types, including neurons[15]?

IS THERE A POSSIBLE HIERARCHY AND ROLE AMONG MILK STEM CELLS?

An important question emerged from many studies focused on a possible hierarchy related to different roles of these maternal milk associated stem cells: What is the reason behind the presence of different patterns of all these cells in maternal milk? What advantages do breast-fed infants gain from receiving huge amounts of living cells every day of their postnatal life? The potential benefits of milk cells were clearly defined by Hassiotou and Geddes[16], a researcher who has played a major role in topic of this field of human physiology. According to Hassiotou and Geddes[16], the maternal cell pool should be subdivided into two main pools with different functions.

The first pool, which includes immune cells such as lymphocytes and monocytes, is thought to be involved in providing important immediate immunological support to the newborn, protecting the breast-fed infant from infections in the perinatal period. The relevance of this maternal milk cell pool has been confirmed by more recent studies focused on the innate immune system of human milk[17,18]. According to this vision, immune cells should be considered the cells given by the mother to the infant for her or his survival, providing the newborn with relevant protection against the large number of microbes to which the infant is exposed during delivery[19]. The second pool of plastic stem/progenitor cells should be considered to be involved in the postnatal development of breast-fed infants, with short-term and long-term consequences for newborn life and health status later in life[14,16].

This revolutionary hypothesis was accepted with caution by the scientific community, with some papers underlying the mystery of stem cells in milk[20]. For other authors, the presence of stem cells in human milk was identified as a new fascinating challenge for perinatologists[21]. Surely, the discovery of a high number of maternal stem cells in the breast milk opened a new chapter in foetal programming theory. Barker et al[22] hypothesis noted the ability of factors that act in the prenatal and perinatal periods to influence susceptibility to developing multiple diseases later in life. According to Barker et al[22] theory, breastfeeding should be included among the multiple factors available to shape susceptibility, or alternatively resistance, to the insurgence of diseases later in life, including those encountered in childhood or adulthood. This suggestion should be verified by further studies aimed at verifying the role of breastfeeding as a protective agent against the development of chronic diseases such as neurodegenerative disorders[23,24].

CAN MILK STEM CELLS ENTER NEWBORN CIRCULATION?

The second question concerns the ability of these stem/progenitor cells to cross the intestinal barrier and enter newborn blood. The first problem to address was the survival of stem cells in the stomach of the neonate. In contrast to adult subjects, the newborn stomach at birth is characterized by a mean pH: 5.28, with pH values > 4.0 in the first week of postnatal life[25]. Moreover, in breastfed infants, the tendency of the gastric pH towards neutral values is sustained by the buffering capacity of milk. These simple considerations suggest that maternal milk stem cells can survive during gastrointestinal transit.

Accepting that maternal stem cells may survive during gastric barrier transit, what is their destiny in the newborn gut? Hassiotou et al[26]and coworkers organized a very elegant experiment in mice that provided an astonishing answer to this question. After selecting mice with cells that express terminal deoxynucleotidyl transferase (TdT) and mice not expressing this antigen, TdT-positive mothers were induced to breastfeed newborn mice born to TdT-negative mothers. Following breastfeeding from TdT-positive dams, TdT-negative newborn mice initially exhibited chimerism, with TdT-positive cells found in the stomach wall, portal vein blood, and liver. This study provides the first evidence of the migration and integration of maternal stem cells inside various organs of lactating neonates.

Another experiment performed in murine pups confirmed that breast milk leucocytes may survive in suckling infants. Most of these cells are established in in Peyer’s patches of the neonatal intestine, where maternal myeloid precursors differentiate into cytotoxic T cells[27]. On the basis of the ability of breast stem cells to differentiate into neurons and glial cells in vitro, the potential of stem cells to migrate into the brain and differentiate into neurons and glial cells, facilitating newborn brain development, was hypothesized[14,28].

THE DARK SIDE OF THE COIN: CHEMICAL SUBSTANCES IN BREAST MILK AND STEM CELLS

We present 2 examples: Endocrine disruptors and antibiotics.

Endocrine disruptors

Endocrine disruptors are chemicals that can alter the normal functioning of the endocrine system. These compounds are present in many everyday products, such as pesticides, plasticizers, detergents, cosmetics and foods, and can have negative effects on human health, especially during prenatal and neonatal development. Endocrine disruptors can be transferred from mother to newborn through breast milk. Exposure to these substances can have long-term consequences for a child’s health, affecting the development of the endocrine system and other body systems. Some studies have shown that exposure to endocrine disruptors can alter the composition of breast milk, including stem cell number and function. In fact, the available literature demonstrates that EDCs can influence the biology of stem cells in a variety of ways, for example, by altering hormonal pathways, DNA damage, epigenetic changes, reactive oxygen species production and gene expression patterns[29]. In particular, endocrine disruptors can interfere with the development of the endocrine system, with long-term impacts on metabolic disorders, neurodegenerative diseases and reproductive health.

In a systematic review that examined 50 scientific publications (over 3000 samples of breast milk), bisphenols, parabens, and benzophenones were detected in approximately half of the breast milk samples[30]. The influence of endocrine disruptors on stem cells present in breast milk, which are essential for tissue regeneration and growth, could reduce the effectiveness of breast milk in supporting the development of the newborn. In fact, research on the interaction between endocrine disruptors and stem cells in breast are ongoing. Some studies have explored their potential implications. Breast milk contains stem cells and bioactive components that can be affected by endocrine disruptors, potentially altering their regenerative potential[31]. These studies indicate that endocrine disruptors may have adverse effects on various types of stem cells. However, specific research on the direct effects on stem cells in breast milk is still limited. It is therefore very important to limit mothers’ exposure to these substances, especially during pregnancy and breastfeeding.

Antibiotics

With respect to antibiotics, such drugs can negatively impact gut microbiota development in infants[32]. Most studies have focused on the administration of antibiotics directly to infants or prenatal exposure in utero. However, clinical data on one of the most common routes of infant antibiotic exposure, i.e., human milk, are limited. The transfer of drugs to infants via human milk is a critical research topic that needs further researcher. Historically, lactating parents have been routinely excluded from clinical research for different reasons, including ethical and safety concerns[33]. Cox et al[34] demonstrated that early destruction of the original composition of the intestinal microbiota using antibiotics can lead to long-lasting alterations in the composition and metabolic activity of the microbiota itself, resulting in adult-onset obesity. These results show that even transient perturbations in the gut microbiota during critical time periods of development can have long-term effects on the host. The same scholar also hypothesized that the negative effects of antibiotics could manifest themselves through negative effects on stem cells[34].

We know that breast milk stem cells migrate from the newborn’s intestine into various organs of the newborn itself, particularly into the brain, where they transform into neurons, astrocytes, and oligodendrocytes[35,36]. The presence of potentially harmful and neurotoxic chemical substances in breast milk must be assessed both in terms of associations with neurodevelopmental disorders in childhood, particularly in the first thousand days after conception, and in terms of predisposing individuals to long-term neurological diseases, such as Parkinson’s disease or Alzheimer’s syndrome. These data do not diminish the fact that breast milk is an extraordinary liquid. However, awareness to defend against the harmful effects of chemicals and environmental contaminants must significantly increase. Moreover, it must also be emphasized that breast milk-derived mesenchymal stem cells have shown significant potential in mitigating neurological damage induced by substances such as toluene in rats, suggesting that breast milk could have significant therapeutic properties even in the presence of toxic substances[37].

CONCLUSION

There is much to learn regarding the multiple subtypes of stem cells present in human breast milk and their physiological and clinical potential[38]. Recent studies focused on the stem cell pool have revealed new insights into the complex relationships between mothers and newborns in terms of cell transfer from mother to the foetus during gestation and through breastfeeding during the perinatal period. Two independent pathways of maternal cell transmission have been identified: Through the placenta during pregnancy and through breastfeeding after birth[39]. One of the most important findings concerning milk stem cells is that they are alive when consumed by newborns. As a consequence, these cells may have beneficial effects on the breastfed infant. According to some authors, stem/progenitor cells found in maternal milk might operate as “developmental training” for nursing infants, resulting in the prevention of type II diabetes, neurodegenerative diseases and kidney disease later in life[40].

Taken together, these findings reinforce the concept of the exclusivity of maternal breast feeding for the best development of any neonate. Moreover, the ability of stem/progenitor cells to survive in the gut of breast-fed newborns and differentiate into different cell lines when they enter the organs of neonates may open a new field in neonatology that could potentially be termed “Developmental Neonatology”[7]. The presence of the ISL1 pioneer transcription factor in some human breastmilk cells (Figure 3) confirms that specific cell lineage epigenome messages could have important effects during postnatal growth[14].

From this perspective, a recent study on the cellular components of the colostrum and milk of preterm mothers revealed that preterm mothers’ milk has increased levels of haematopoietic and mesenchymal stem cells, suggesting that developmental training might be even more important in preterm newborns. The hypothesis that maternal breastfeeding could represent an important factor for the long-term growth and health status of preterm babies should be verified in further studies[41]. On the one hand, breast milk is an extraordinary liquid; however, awareness of its ability to defend against chemicals and environmental contaminants must significantly increase.

Future perspectives

The presence of many stem/progenitor cells, which are able to differentiate into many different cell types, in the breast milk and their availability at a very low cost without the need to obtain complex permissions for their use has prompted multiple authors to consider the use of maternal milk stem cells in clinical practice in recent years. The possible fields of application of milk stem cells are numerous, including all fields of regenerative medicine. A recent reported an innovative approach, namely, the intranasal parent’s own milk (IPOM) technique[42]. In this study, 37 preterm infants affected by intraventricular hemorrhage were received maternal breast milk via intranasal injection. The objective of this study was to favor the passage of maternal stem cells through the permeable neonatal blood-brain barrier, allowing them to home to the neonatal brain, followed by their differentiation into neuronal and glial cells. In phase 1, the study showed that IPOM is well tolerated by newborns, demonstrating the feasibility of this approach. Further studies will explore the value of IPOM in the clinical development of preterm babies affected by intraventricular hemorrhage. Extensive research is needed to characterize milk stem cell pools, both at term and in preterm newborns, with a focus on the multiple potential uses of breast milk stems/progenitors in regenerative therapies in preterm neonates. Figure 1 graphically shows the possible correlations between different milk stemness patterns and an increased predisposition to future diseases (Barker’s theory).