Stem and Progenitor Cells in the Retina

Abstract

Regardless of the debate regarding moral issues of using stem cells in research, they are unequivocally useful for understanding pathological angiogenesis, particularly so in the retina. Some important stem cell concepts include a niche, as well as the ideas of self-renewal and plasticity. Self-renewal is the maintenance of a stem cell population, through production of both undifferentiated and further differentiated cells (precursors), while plasticity is the differentiation of a stem cell into various cell types. However, questions regarding plasticity exist, since cell fusion was shown to be the underlying cause for some plasticity observations. Well-studied types of stem cells include neural stem cells, mesenchymal stem cells, hematopoietic stem cells or progenitors such as endothelial precursor cells. Different cell surface markers help classify these cells types. Hematopoietic stem cells and endothelial precursor cells are involved in angiogenesis. Numerous hypoxia-regulated factors have been implicated in angiogenesis, including vascular endothelial growth factor, stromal derived factor-1, insulin-like growth factor, and monocyte chemoattractant protein-1. Progenitor cells, found amongst both early (CD34+) or late (CD14+) blood mononuclear cells, are impaired in diabetes. Studying these types of cells, along with others, can dissect the precise molecular mechanisms underlying stem/progenitor cell activity in the retina.

Copyright © 2010 S. Karger AG, Basel

Despite the ongoing debate about the ethical considerations regarding stem cell use, there is

no denying that they are an extremely useful scientific tool for understanding disease and repair processes [1]. Normally, stem cells restore function when there is cell loss due to turnover or damage. The lineage specification of a particular stem cell depends largely on its environment.

There are three mammalian pluripotent embryonic stem (ES) cell lines that have been isolated; they are the embryonal carcinoma, ES and embryonic germ cells [2]. In the adult, the bone marrow (BM) contains the greatest number of stem cells. There are several types of stem cells present, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and additional nonhematopoietic cells [3]. The use and manipulation of BM stem cells, such as for therapeutic purposes, requires a better understanding of work characterizing the cell populations and their functions [3].

Niches

A niche is regarded as tissue or extracellular matrix (ECM) that can support the existence of at least one stem cell. In stem cell biology, continuous debate exists on whether or not niches exist and are necessary for stem cells to maintain their

characteristics. The most compelling evidence for their existence comes from studies on spermatogenesis, in which germ cells are maintained by specialized stem cells and when the germ cells begin to divide, detach from the basement membrane [4, 5]. The niche also functions to control self-renewal and daughter cell production rates. Often niches function to keep pace with growth from youth to adulthood. In a lineage niche, the number of cells does not change over time. The stem cell divides in such a way that only one daughter cell is preserved in the niche as a stem cell. In the population niche, the fate of cells is somewhat less certain. Both daughter cells may continue to be stem cells or both may differentiate [6].

Characteristics of a Stem Cell

Although the exact definition of a stem cell is difficult to determine, it is generally agreed that stem cells must be capable of self-renewal and plasticity. Stem cells are usually depicted in a hierarchy of differentiation capacity; the totipotent fertilized egg is the ultimate stem cell, resulting in every differentiated cell by way of pluripotent ES cells. ES cells then lead to multipotent adult stem cells, which then give rise to non-self-renewing oligopotent progenitor and or precursor cells (fig. 1). In the hematopoietic lineage, for example, the adult stem cell gives rise to a common myeloid and a common lymphoid progenitor.

Self-Renewal

Self-renewal is the maintenance of a stem cell population, usually through asymmetric division of a cell that produces one cell that is more differentiated than the parent and one that retains the undifferentiated stem cell qualities. This definition can also include the continuance of a stem cell pool, not only the same type of stem cell.

Plasticity

There has been intense debate regarding the extent of plasticity of stem cells. Plasticity is a property that includes the differentiation of a stem cell or a stem cell pool into a wide array of cell types. It also includes transdifferentiation from a cell that has become somewhat committed to a particular fate into a cell type that has an entirely different fate. Even more radical is the idea of dedifferentiation, where a cell can become less committed along a particular lineage and display more primitive characteristics [7]. Some evidence suggests that stem cells are quite flexible and can transdifferentiate extensively. For example, it has been shown that adult stem cells can differentiate into cell types that are quite different from the original cell, even crossing over germ layer distinctions [8, 9]. Cells from the BM have been shown to differentiate into an enormous variety of tissue, including muscle [10, 11], neural cells [12–14], hepatocytes [15–19], kidney [20, 21], lungs [22], GI tract [22], skin [22], myocardium [23–25] and blood [26].

On the other hand, some reports question the degree of plasticity in stem cells, even going so far as to question the existence of cell transdifferentiation. One report shows that stem cells from the central nervous system rarely differentiate into blood [27]. Wagers et al. [28] reported that HSCs did not contribute significantly to nonhematopoietic tissues such as muscle, kidney, gut, liver, or brain. This study showed considerable hematopoietic contribution to blood, but rarely outside of the blood compartment. Only one other cell type studied, a Purkinje cerebellar neuron, was identified as having derived from the HSC. This cell type was also the only identified cell type in two other similar studies [29–31].

Fusion

Cell fusion events have been shown to be at least partially responsible for some cell plasticity observations. When neurosphere-derived cells were cocultured with ES cells, the cells fused [32].

Fused cells were also reported by Terada et al. [33] using ES and BM cells. These cells were tetraploid, had the properties of ES and displayed markers of both of the parent cell types. These fused cells were found in the liver, intestine, kidney and heart [33]. Fusion events were also reported in a study involving heat-shocked small airway epithelial cells and human MSCs [34].

Although these fused cells have been seen in more than one study, it is unlikely that they are

responsible for the results in all ‘transdifferentiation’ studies. Fusion is still seen as a relatively rare event. To conclude that fusion has indeed occurred, a study must show evidence of increased chromosome number, as well as the presence of both cell types. It is also important to remember that cell processes that occur in normal animals can be quite different and occur at a different rate from those that occur in injured animals.

Types of Stem and Precursor Cells

Neural Stem Cells

Neural stem cells (NSCs) are progenitor cells that can be isolated from the central nervous system, peripheral nervous system and the embryonic nervous system that can give rise to neurons and glia [35–40]. Based on these findings, it is possible that what was considered an inflexible tissue could have regenerative ability [41]. Cell surface markers that define NSCs have not yet been compiled; rather NSCs are classified based on the cell types they produce. Cultured pluripotent embryonic stem cell can produce a type of NSC, but only approximately 0.2% of embryonic stem cells produce neurospheres [42]. Stem cells have even been found in the ciliary margin of the adult mammalian retina [43].

Mesenchymal Stem Cells

MSCs, also called stromal or skeletal stem cells, are found in the BM and have been thought to differentiate into several cell types, including bone, cartilage, fat, muscle, marrow stroma and tendon [10, 44–49]. When isolated from an adult human, the cells could be stimulated to produce adipocytic, chondrogenic, or osteogenic cells [50]. Compiling the cell surface markers to identify MSCs has proven difficult, partially due to cross-reactivity of markers with other cell types [51, 52]. A distinguishable characteristic of MSCs is their adherence to tissue culture plastic [8].

Work presented by Verfaillie et al. [53] suggests MSCs differentiate into endothelium, liver and neural cells and may be able to differentiate into all cell types [31]. A type of BM-derived cell termed the multipotent adult progenitor cell was shown to co-purify with MSC. These cells were shown to be CD34–, VE-cadherin–, AC133+ and Flk1+, signifying that they were nonendothelial in nature. However, when these cells were cultured in the presence of vascular endothelial growth factor (VEGF), the cells differentiated into cells that express known endothelial markers. In addition,

the cells played a role in tumor angiogenesis as well as wound healing. These cells may provide a novel source of endothelial cells for future therapies [53]. The same authors have shown that these multipotent adult progenitor cells differentiate into cells that have mesodermal, neuroectodermal and endodermal characteristics in vitro, and differentiated into cells of the hematopoietic and epithelial lineages in vivo [54].

Hematopoietic Stem Cell

HSCs are believed to be the cells that, besides assisting hematopoiesis, also would drive adult vasculogenesis. They are defined by the ability to differentiate into all cells of the vascular system. The close location of endothelial cells and hematopoietic cells in the early embryonic blood islands has indicated a common ancestor cell, the hemangioblasts [55]. Further evidence for this common ancestor cell is that both lineages display common cell surface markers, such as Flk-1, Tie 2, CD34 and SCL/TAL [56–60]. HSCs home rapidly through the blood to the BM [61]. In vitro studies have shown that when HSCs are in contact with stromal cells, their proliferation rate increases [62, 63]. HSCs may vary in their capacity to self-renew. Krause et al. [22] have shown that one HSC could differentiate into cells of endodermal and ectodermal organs. Multipotent progenitors comprise approximately 0.05% of mouse BM cells. Within this cell population, three groups exist, the long-term and the short-term self-renewing HSCs and the multipotent progenitors that do not have measurable self-renewing capacity [64].

Several studies have shown that recruitment and subsequent differentiation of HSCs to sites of mechanical injury in the retina contribute to retinal neovascularization in a murine model [26, 65–68].

It is still relatively unknown exactly which genes are responsible for HSCs to retain their differentiation characteristics. Early on, it was widely believed that the stem cell leukemia/tal-1 gene

was required for HSCs. However, this notion was placed into question by Mikkola et al. [69] when it was concluded that this gene was seemingly required for the initial generation of HSCs, but not for the retention of HSC characteristics.

Endothelial Precursor Cells

During development, HSCs are found in the center of blood islands, whereas the endothelial precursor cells (EPCs) are found along the periphery. In 1991, George et al. [70] unequivocally demonstrated the presence of circulating endothelial cells (CECs) in whole blood using an endothelial cell-specific antibody. Since that time, a number of different laboratories have identified CECs in whole blood by the use of endothelial cell-specific monoclonal antibodies and cell culture in a variety of pathologic conditions. In normal individuals, there are approximately 0–20 CEC per milliliter of blood. CECs may be derived from two sources: shed from the vasculature or, more interestingly, released from the BM. Cells derived from the vasculature would be mature endothelial cells and express phenotypic endothelial cell markers such as von Willebrand factor, VE-cadherin, CD146, or TE-7. These mature endothelial cells may detach due to mechanical disruption [71, 72]. If CECs originate from the BM, they are derived from EPCs and can fully differentiate to endothelial cells, expressing mature endothelial cell markers. Unfortunately, few studies have addressed these hypotheses to determine the true origin of CECs [73, 74].

Although it is not clear what markers precisely define an EPC, it is clear that cells derived from the BM will populate an area of neoangiogenesis. In a neovascular mouse model, 8–11% of the endothelial cells were of EPC origin, whereas hematopoietic progenitors populate about 2% of the vasculature in stable adult tissue [75]. Similar results are seen in the neovascularization that occurs in the endometrium during ovulation and wound healing in mice [76]. There are both circulating HSCs and EPCs that have the capacity

to populate both the BM and neovasculature [61]. Although HSCs are generally thought to be the ancestor of the EPCs, it has been shown that MSCs can also differentiate into EPCs [22].

Several studies have indirectly addressed the issue of the presence of EPCs in the circulation and their role in postnatal vasculogenesis. The progenitor cell markers CD133 or CD34 are seen on the EPCs. After 7 days of culture on fibronectin, CD34+ mononuclear cells display an endothelial cell phenotype, are able to incorporate acetylated low-density lipoprotein, produce nitric oxide when stimulated with VEGF and express platelet/endothelial cell adhesion mole- cule-1 and Tie-2 receptor [77]. It is also believed that a unique subset of cells expressing CD133, CD34 and VEGFR-2 may be an additional source of EPC [78, 79]. Cells that express both CD133 and CD34 are believed to be more primitive EPCs, whereas CD133– but CD34+, VEGFR-2+ cells may represent a more mature, differentiated population of EPCs [78, 79]. In support of this, CD34+ cells enriched for CD133+ cells do not express VE-cadherin or von Willebrand factor and only 3% of these cells express VEGFR-2. However, after 3 weeks of culture and further purification with Ulex europaeus agglutinin (a lectin-recog- nizing endothelial cells), cells expressed several specific endothelial markers (von Willebrand factor, CD146, CD105, E-selectin, VCAM-1 and VE-cadherin) [80]. Several studies have used animal models to examine neovascular development in response to exogenous administration of various agents as well as targeted mutations [81, 82].

Bone Marrow-Derived Cells Participate in

Normal Maintenance and Repair of the

Endothelium

As mentioned earlier, circulating BM-derived cells participate in normal maintenance of the endothelium [75, 83–87]. Approximately 1–12% of endothelial cells in blood vessels are BM derived;