Reproductive Stem Cell Differentiation

The ECM or Tissue Microenvironment Directs MSCs Lineage Specification

It has already been shown that MSCs are frequently recruited to the sites of tissue injury or inflammation and differentiate into multiple specific lineages, ^27,35,43 but the mechanisms underlying this process are not clear. The ECM elasticity or stiffness seems to be vital for cell lineage specification.

According to the matrix elasticity, tissues can be classified into 3 distinct groups: (1) softer tissues as the brain, (2) stiff tissues as the muscles, and (3) rigid tissues as the bones. ^{44 –46} It is believed that the great variation in tissue or ECM microenvironment provides specific conditions that guide cell differentiation. In this context, the molecular mechanism underlying the MSCs interaction with the tissue or matrix and how it will transduce those stimuli in cell morphological modification are not yet well understood. ⁴⁵

Studies have been carried out mostly evaluating the contribution of ECM microenvironment in chondrogenesis, osteogenesis, and adipogenesis, ^{41 ,45 –48} and in cancer development. ⁴⁹ In addition, Masuda et al ² studied the effects of local environment in the endometrial regeneration by stem cells.

It has been hypothesized that during cell–matrix interaction, specific stimuli are produced and are transduced in chemical signaling events for distinct phenotype expression. ^{45,50 –52} In their niche or within tissue, cells are exposed to mechanical forces. When cells are submitted to these forces, a variety of physiological modifications occur such as cell motility, proliferation, and differentiation. ⁵³ The mechanisms by which the cells recognize these mechanical perturbations and transduce them in specific chemical signaling remain obscure. ^50,52 It is known that the matrix stiffness or elasticity plays a key role in cell lineage commitment ^32,47,48 and influences focal-adhesion structure and the cytoskeleton. ⁴⁵

The development of extracellular matrix models with variable degrees of elasticity based on inert polyacrylamide gels mimicking the consistency of the different tissues is necessary in order to evaluate the differentiation of MSCs. ⁴⁵ They observed that in soft matrices, that mimics brain tissue, the MSCs showed a neuronal phenotype, in intermediate stiffness matrices that mimics striated muscle, the same cells differentiated into myoblast, and in rigid matrices resembling cartilage or bone, the cells became osteoblasts. This study provided great evidence that the matrix composition can drive cell differentiation. ⁴⁵

Two key events are present in the described processes, the ability of the MSCs to recognize the extracellular matrix forces and, second, the mechanic stimuli transduction to generate specific signals that will drive the differentiation. ⁴⁵ Multiple signaling pathways are activated in response to mechanical force stimulation. McBeath et al ⁴¹ observed that both matrix stiffness and soluble factors modulate MSC-specific lineage commitment via RhoA signaling and Rho-kinase (ROCK) activity, which regulates the actin–myosin contractility. It has been shown that inhibition of cytoplasmic myosin II using blebbistatin disrupts the influence of matrix elasticity in cell differentiation. ^45,46 These findings suggested that during force transduction, specific signaling pathways are activated including Rho guanosine triphosphatase, mitogen-activated protein kinase, tyrosine kinases/phosphatases, ^49,52,54 which can alter gene expression and promote cell differentiation. ^{41,45,46,55,56}

Comparative studies of microenvironment and growth factors’ influence in MSCs differentiation were performed. Davis ⁵⁶ investigating the influence of both bone morphogenetic protein 2 and three-dimension (3D) osteoconductive substrates in osteogenesis, observed enhanced effect in osteogenic response of MSCs due to multiple pathway activation mediated on both substrate and growth factor.

The ECM microenvironment seems to be more selective than other factors and can alter the phenotype in precommitted cells. ⁴⁵ Although satisfactory in vitro results has been achieved in vitro, many molecular issues underlying the cell commitment must be clarified for future application in vivo.

Another key point in cell investigation is related to cell medium for culture. Many conclusions on ECMs culture influencing cell lineage commitment derive from studies on culture of cells in two dimension (2D) surfaces. A different behavior have been described when the cell culture is done on 3D approaches. ^46,53,57,58 Notwithstanding, it was shown that cell types derived from in vivo settings quickly lose their differentiated phenotype when are plated onto 2D medium. ⁵⁸ Besides, in vitro differentiation studies relies on biological intervention, like special media but scaffolds are important to avoid possible host reactions in vivo. ⁵⁹

Recently, Jurgens ⁶⁰ induced chondrogenesis in a collagen type II hydrogels mimicking the joint 3D microenvironment, Schneider et al ⁴⁴ induced tenogenic differentiation in 3D high-density microenvironment, and Mohr et al ¹³ produced an osteogenic graft in a chorion-derived scaffold from MSC derived from human placenta. Together, these results demonstrated that the ECM stiffness or elasticity play a key role in cell differentiation. ^46,47,49

The Effect of Soluble Growth Factors in Cell Differentiation

As was reported in several studies in vitro, the cell differentiation involves numerous extra and intracellular signaling pathways modulated by either cell–cell or cell–matrix contact as well as soluble growth factors. ² The growth factors are peptides that can be produced by the target cell (autocrine) or released through the plasma membrane of adjacent cells (paracrine), modulating cellular activity. ⁶¹

The effects of several different growth factors in MSC lineage specification was extensively studied in vitro in chondrogenesis, ^{62 –64} osteogenesis, ^{64 –68} and adipogenesis. ^{69 –71} Many growth factors promoting MSCs proliferation and differentiation have been described, but the exact molecular mechanisms by which they act is still unclear. To better understand these mechanisms, it is first necessary to identify the factors that promote proliferation, differentiation, or retain the differentiation state of MSCs. ²⁴

The influence of growth factor in cell differentiation varies not only in stem cell type but also in the strain that is sought. Many factors have been described as MSC differentiation inducers including platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF) β, epidermal growth factor (EGF), and insulin-like growth factor (IGF). ^{27,72 –74} These factors were previously described by Kuznetsov et al ⁷⁵ as essential for bone marrow stromal fibroblast colony formation in vitro. Beyond the cell differentiation, they also modulate the angiogenesis, ECM composition, chemotaxis of osteoprogenitor cells, and granulation or scar tissue deposition. ^65,74 Recent studies showed the importance of stromal-epithelial cell interactions in the endometrial epithelial stem cell niche. They identified several growth factors, such as EGF or TGF-α or PDGF-BB , and fibroblast feeder, layers to establish clonal growth. ^76,77

Subsequent studies ^{62 –65,68 –71} have found wide variation in MSCs response to growth factors in different cell lineage specification. Although the endometrial differentiation has been already described, ^3,78 the factors involved in this process need to be further studied. Different factors can be required for cell specification depending on the stimuli (Table 1).

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Table 1.

Wide Variation of Growth Factors Involved in MSCs Differentiation. Growth Factors Involved in MSCs Differentiation.^a

MSCs differentiation	Growth factors	References
Chondrogenic differentiation	BMP2 + TGFβ	Schmitt, et al 200362
	bFGF	Davidson, et al 2005 63
		Cuevas, et al 1988 79
	TGF-β3	Mackay, et al 1998 80
Osteogenic differentiation	BMP	Chen, et al (1998) 81
		Davis et al 2011 56
		Yonezawa et al 2011 82
	BMP1-3	Grgurevic et al 2011 83
	BMP2 + bFGF	Hanada, et al 1997 65
	bFGF	Hanada, et al 1997 65
	TGF- β1	Macdonald et al 2007 73
	bFGF + PDGF-B	Fierro et al 2011 67
	BMP + TGF- β	Gorter et al 2011 68
	HGF	Wen et al 2011 84
Adipogenic differentiation	bFGF	Neubauer et al 2004 70
	FGF-2	Kakudo et al 2007 71
Tenocytes-Like differentiation	IGF-1 + TGF- β1	Schneider et al 2011 44
Cardiomyogenic differentiantion	NPY	Wang et al 2010 85
Neuron-Like differentiation	EGF + HGF + VEGF	Bae et al 2011 28
	EGF + bFGF	Delcroix et al 2010 86
Hepatogenic differentiation	HGF	Pulavendran et al 2011 87
Endometrial differentiation	E2	Kulak et al 2012 78

Abbreviation: BMP, bone morphogenetic protein; TGF, transforming growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; NPY, neuropeptide Y; HGF, hepatocyte growth factor; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor; PDGF, Platelet-derived growth factor; MSCs, mesenchymal cells.

^a The exact influence of each grow factor in MSCs differentiation is not clear due the complexity mechanism and interactions among these factors. Depending on the culture medium where the cells are inserted, a certain growth factor can induce multiple lineages differentiation.

The exact effect of each growth factor in cell differentiation is not clear because the mechanism underlying this process is complex. For example, it was observed in vitro that depending on the culture medium where the cells are inserted, a certain growth factor can induce chondrogenic, osteogenic, or adipogenic differentiation. It has been shown that FGFs are crucial for chondrogenesis, ^63,79 adypogenesis, ^70,71 and osteogenesis. ¹⁶ On the other hand, chondrogenic differentiation was observed to be inducible by bone morphogenetic protein (BMP) ^62,64 or TGF and dexametazone. ⁸⁰ It was believed that BMPs was only fundamental for upregulation of cartilage and bone phenotype expression. However, it have been demonstrated that BMP are also implicated in several cell type grow and specification. Specific lineages were obtained in several conditioned medium with BMPs including, bone, cartilage, fat, and nervous cells. ⁶² Interactions between one or more factors result in enhanced effect in MSCs differentiation. Hanada et al ⁶⁵ and Deans and Moseley ²⁴ reported that bFGF and BMP-2 synergistically enhanced the osteogenic differentiation of MSCs in vivo.

Basically, the growth factors interact with distinct cell receptors activating different intracellular signaling pathways which turn on transcriptional factors for specific gene expression depending on the stimuli.

Chen et al ⁸¹ reported that selective activation of BMP receptor type 1B (BMPR-1B) result in differentiation of MSC to osteoblastic lineage, whereas activating the BMBR-1A these cells differentiate into adipocyte lineage. This latter information was suggestive that the overexpression or loss of the receptors may be important in determining the response of MSCs to growth factor stimulation. ²⁴ Most recently, Lin and Hankenson ¹⁶ studied the interactions of BMP, Wnt, Notch, hedgehog, and FGF signaling pathways in osteoblastogenesis. In all signaling pathways, the transcriptional factor Runx2 is activated and seems to play a key role in osteogenic differentiation. However, the more precise molecular understanding of these complex interactions is recommended.