6.3.1. Scanning electron microscope (SEM)

The two-dimensional morphology of MSCs demonstrated by scanning electron microscope (SEM) [38] showed the spindle-shaped cells with eccentric nuclei and several thin cytoplasmic processes extending from the edge of the cell surface in P5 and P9. In addition, cells in P 9 maintained their spindle shape (Figure 4). These SEM results were also reported [49].

Figure 2. Cultured human bone marrow derived stromal cell from passage 5, showing adherent cells with their characteristic spindle shape (arrow) [38]. Scale bar 200 μm.

Figure 3. Cultured human bone marrow derived stromal cell from passage 9, showing adherent cells with their characteristic spindle shape (arrow) [38]. Scale bar 200 μm.

#### 6.3.2. Transmission electron microscope (TEM)

Electron microscopic examination of MSCs in culture revealed the presence of euchromatic nucleus associated with abundant cell organelles which are considered as an indicator of an active cell (Figure 5). The spindle-shaped cells showed large irregular, euchromatic nucleus and the peripheral heterochromatin was slightly condensed along the inner surface of the nuclear membrane and nuclear pores (Figure 6). The cytoplasm showed many elongated profiles of rough endoplasmic reticulum and multiple mitochondria (Figure 7). Cytoskeletal structures were seen as fine filaments running parallel to the long axis of the cell near the nuclear membrane as well as beneath the cell membrane (Figure 6).

The same features of active MSCs were noticed after 14 days in culture. The cells exhibited a large euchromatic nucleus with numerous profiles of rough endoplasmic reticulum and multiple rounded mitochondria. In addition, the cell surface showed thin pseudopodia (Figure 7). Cytoskeletal filaments were irregularly dispersed in the cytoplasm as well as around the nucleus (Figure 7). Such observation was explained by the fact that the intracellular organelles architecture is organized by the cytoskeleton [36, 50, 51].

Figure 4. Cultured human bone marrow derived stromal cell, showing spindle shape cell with an eccentric nucleus (N) and multiple processes (P) [38]. Scale bar 50 μm.

> Moreover, after 21 days in culture, the cells showed clearly demarcated nucleolus (Figure 8). In addition, numerous large macro vesicles associated with the mature face of the Golgi complex were clearly depicted (Figure 9). These cells are now ready for differentiation once in the appropriate media. The structure of these cells would differ during the process of differentia-

> Figure 7. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 14. The cytoplasm exhibits numerous profiles of rER, mitochondria (M), and well-developed Golgi complex (G). The cell membrane exhibits a pseudopodium (Pd). Cytoskeletal filaments are irregularly dispersed in the cytoplasm (arrows). Part of

> Figure 6. Transmission electron micrograph of cultured human bone marrow derived stromal cell showing part of the same cell exhibiting an euchromatic nucleus (N) with nuclear pores (arrow heads). The peripheral heterochromatin (H) is seen along the inner aspect of the nuclear membrane. Fine cytoskeletal filaments are noticed parallel to the long axis of the

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cell near the nuclear and cell membranes (arrows) [38]. Scale bar 0.5 μm.

an euchromatic nucleus is also seen (N) [38]. Scale bar 0.5 μm.

Another ultrastructure feature of MSCs is the presence of vesicles in the cytoplasm. Intercellular communication can be mediated through direct cell–cell contact or transfer of secreted molecules. Recently, a third mechanism has emerged that involves intercellular transfer of extracellular vesicles. Cells release into the extracellular environment membrane vesicles either of endosomal origin or of plasma membrane origin. They are named exosomes and microvesicles, respectively [52]. In the study [38] carried out on isolated MSCs, showed vesicular trafficking. (Figure 10) These vesicles were prominent after the cells were cultured in a

tion accordingly.

Figure 5. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 7. The cell is spindle in shape with an euchromatic nucleus (N). The cytoplasm shows mitochondria (M) and multiple lysosomes (L) [38]. Scale bar 1 μm.

6.3.2. Transmission electron microscope (TEM)

34 Stromal Cells - Structure, Function, and Therapeutic Implications

nuclear membrane as well as beneath the cell membrane (Figure 6).

architecture is organized by the cytoskeleton [36, 50, 51].

and multiple processes (P) [38]. Scale bar 50 μm.

[38]. Scale bar 1 μm.

Electron microscopic examination of MSCs in culture revealed the presence of euchromatic nucleus associated with abundant cell organelles which are considered as an indicator of an active cell (Figure 5). The spindle-shaped cells showed large irregular, euchromatic nucleus and the peripheral heterochromatin was slightly condensed along the inner surface of the nuclear membrane and nuclear pores (Figure 6). The cytoplasm showed many elongated profiles of rough endoplasmic reticulum and multiple mitochondria (Figure 7). Cytoskeletal structures were seen as fine filaments running parallel to the long axis of the cell near the

The same features of active MSCs were noticed after 14 days in culture. The cells exhibited a large euchromatic nucleus with numerous profiles of rough endoplasmic reticulum and multiple rounded mitochondria. In addition, the cell surface showed thin pseudopodia (Figure 7). Cytoskeletal filaments were irregularly dispersed in the cytoplasm as well as around the nucleus (Figure 7). Such observation was explained by the fact that the intracellular organelles

Figure 4. Cultured human bone marrow derived stromal cell, showing spindle shape cell with an eccentric nucleus (N)

Figure 5. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 7. The cell is spindle in shape with an euchromatic nucleus (N). The cytoplasm shows mitochondria (M) and multiple lysosomes (L) Figure 6. Transmission electron micrograph of cultured human bone marrow derived stromal cell showing part of the same cell exhibiting an euchromatic nucleus (N) with nuclear pores (arrow heads). The peripheral heterochromatin (H) is seen along the inner aspect of the nuclear membrane. Fine cytoskeletal filaments are noticed parallel to the long axis of the cell near the nuclear and cell membranes (arrows) [38]. Scale bar 0.5 μm.

Figure 7. Transmission electron micrograph of cultured human bone marrow derived stromal cell on day 14. The cytoplasm exhibits numerous profiles of rER, mitochondria (M), and well-developed Golgi complex (G). The cell membrane exhibits a pseudopodium (Pd). Cytoskeletal filaments are irregularly dispersed in the cytoplasm (arrows). Part of an euchromatic nucleus is also seen (N) [38]. Scale bar 0.5 μm.

Moreover, after 21 days in culture, the cells showed clearly demarcated nucleolus (Figure 8). In addition, numerous large macro vesicles associated with the mature face of the Golgi complex were clearly depicted (Figure 9). These cells are now ready for differentiation once in the appropriate media. The structure of these cells would differ during the process of differentiation accordingly.

Another ultrastructure feature of MSCs is the presence of vesicles in the cytoplasm. Intercellular communication can be mediated through direct cell–cell contact or transfer of secreted molecules. Recently, a third mechanism has emerged that involves intercellular transfer of extracellular vesicles. Cells release into the extracellular environment membrane vesicles either of endosomal origin or of plasma membrane origin. They are named exosomes and microvesicles, respectively [52]. In the study [38] carried out on isolated MSCs, showed vesicular trafficking. (Figure 10) These vesicles were prominent after the cells were cultured in a

media that stimulated its osteogenic differentiation. Microvesicles vary in size and are formed by the budding of the plasma membrane. Most cell types are known to produce microvesicles either constitutively or when stimulated during apoptosis or activation. The mechanisms involved in the mobilization of secretory microvesicles to the cell periphery, their docking, and fusion with the cell surface require the cytoskeleton (actin and microtubules), associated molecular motor proteins (kinesins and myosins) as well as other factors [53, 54]. The other clearly defined class of secreted membrane vesicles that originate from the endosomes are the exosomes. Exosomes were first discovered by Pan and Johnstone in 1983 [55]. They are formed by the invagination of endolysosomal vesicles to form multi-vesicular bodies. Exosomes are released by exocytosis. First, the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed inside the endosome by invaginating parts of the endosome membranes. Such endosomes are called MVBs. Finally, the MVBs fuse with the cell membrane and release the intraluminal endosomal vesicles into the extracellular space to

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http://dx.doi.org/10.5772/intechopen.76870

Exosomes directly interact with the signaling receptors of target cells [57]. After that, the exosomes fuse with the plasma membrane of recipient cells and deliver their content into the cytoplasm [58]. Finally, the exosomes are internalized into the recipient cells. Once in the recipient cell, some of these engulfed exosomes may merge into endosomes and move across the recipient cells to be released into the neighboring cells. In the other case, endosomes fused from engulfed exosomes will mature into lysosomes and undergo degra-

Lipids and proteins are the main components. The protein content of exosomes from different cell types contains different endosome-associated proteins (e.g., RabGTPase, SNAREs, Annexins and flotillin). They are also enriched in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored proteins and flotillin [60]. The other main component of exosomes is the lipid. In comparison to the plasma membrane, exosomes are highly enriched in cholesterol, sphingomyelin and ceramides at the expense of phosphatidylcholine and phosphatidylethanolamine [52]. In addition to the proteins and lipids, various nucleic acids have recently been identified in the exosomal lumen, including mRNAs, microRNAs and other noncoding

The main functions of exosomes are their capacity to act as antigen-presenting vesicles, to stimulate immune responses [62]. Another main important feature of exosomes is being an ideal drug delivery vehicle. Meanwhile, research has been carried out encapsulating anticancer

The function of MSC-derived exosomes has not been well defined. They act as an intercellular communication vehicle for modulating cellular processes. It was recently revealed that exosomes derived from MSCs play important roles in mediating the biological functions of

A study demonstrated the electron microscopy of exosomes. They were cup-shaped and measured 40–100 nm in diameter. Exosomes are naturally secreted and well tolerated by the body. They are also safely stored and provide many therapeutic applications with avoiding the

become exosomes [56].

dation [57, 59].

RNAs [61].

MSCs [64].

drugs into exosomes [63].

Figure 8. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing large euchromatic nucleus (N) with clearly demarcated nucleolus (n). The cytoplasm shows mitochondria (M) [38]. Scale bar 1 μm.

Figure 9. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing part of its cytoplasm with multiple well-developed Golgi complexes (G) associated with large secretory vesicles (V), numerous mitochondria (M), and lysosomes (L). The cytoplasm shows profiles of rough endoplasmic reticulum (rER) [38]. Scale bar 0.5 μm.

Figure 10. Transmission electron micrograph of cultured human bone marrow derived stromal cells. The cytoplasm shows several cytoplasmic vesicles (Vs) of variable sizes. A coated pit (arrowhead) and numerous subplasmalemmal vesicles are also seen (thick arrows). A surface pseudopodium (Pd) is seen [38]. Scale bar 0.5 μm.

media that stimulated its osteogenic differentiation. Microvesicles vary in size and are formed by the budding of the plasma membrane. Most cell types are known to produce microvesicles either constitutively or when stimulated during apoptosis or activation. The mechanisms involved in the mobilization of secretory microvesicles to the cell periphery, their docking, and fusion with the cell surface require the cytoskeleton (actin and microtubules), associated molecular motor proteins (kinesins and myosins) as well as other factors [53, 54]. The other clearly defined class of secreted membrane vesicles that originate from the endosomes are the exosomes. Exosomes were first discovered by Pan and Johnstone in 1983 [55]. They are formed by the invagination of endolysosomal vesicles to form multi-vesicular bodies. Exosomes are released by exocytosis. First, the cell membrane is internalized to produce endosomes. Subsequently, many small vesicles are formed inside the endosome by invaginating parts of the endosome membranes. Such endosomes are called MVBs. Finally, the MVBs fuse with the cell membrane and release the intraluminal endosomal vesicles into the extracellular space to become exosomes [56].

Figure 8. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing large euchromatic nucleus (N) with clearly demarcated nucleolus (n). The cytoplasm shows mitochondria (M) [38]. Scale

Figure 9. Transmission electron micrograph of cultured human bone marrow derived stromal cells on day 21, showing part of its cytoplasm with multiple well-developed Golgi complexes (G) associated with large secretory vesicles (V), numerous mitochondria (M), and lysosomes (L). The cytoplasm shows profiles of rough endoplasmic reticulum (rER)

Figure 10. Transmission electron micrograph of cultured human bone marrow derived stromal cells. The cytoplasm shows several cytoplasmic vesicles (Vs) of variable sizes. A coated pit (arrowhead) and numerous subplasmalemmal

vesicles are also seen (thick arrows). A surface pseudopodium (Pd) is seen [38]. Scale bar 0.5 μm.

bar 1 μm.

36 Stromal Cells - Structure, Function, and Therapeutic Implications

[38]. Scale bar 0.5 μm.

Exosomes directly interact with the signaling receptors of target cells [57]. After that, the exosomes fuse with the plasma membrane of recipient cells and deliver their content into the cytoplasm [58]. Finally, the exosomes are internalized into the recipient cells. Once in the recipient cell, some of these engulfed exosomes may merge into endosomes and move across the recipient cells to be released into the neighboring cells. In the other case, endosomes fused from engulfed exosomes will mature into lysosomes and undergo degradation [57, 59].

Lipids and proteins are the main components. The protein content of exosomes from different cell types contains different endosome-associated proteins (e.g., RabGTPase, SNAREs, Annexins and flotillin). They are also enriched in proteins that associate with lipid rafts, including glycosylphosphatidylinositol-anchored proteins and flotillin [60]. The other main component of exosomes is the lipid. In comparison to the plasma membrane, exosomes are highly enriched in cholesterol, sphingomyelin and ceramides at the expense of phosphatidylcholine and phosphatidylethanolamine [52]. In addition to the proteins and lipids, various nucleic acids have recently been identified in the exosomal lumen, including mRNAs, microRNAs and other noncoding RNAs [61].

The main functions of exosomes are their capacity to act as antigen-presenting vesicles, to stimulate immune responses [62]. Another main important feature of exosomes is being an ideal drug delivery vehicle. Meanwhile, research has been carried out encapsulating anticancer drugs into exosomes [63].

The function of MSC-derived exosomes has not been well defined. They act as an intercellular communication vehicle for modulating cellular processes. It was recently revealed that exosomes derived from MSCs play important roles in mediating the biological functions of MSCs [64].

A study demonstrated the electron microscopy of exosomes. They were cup-shaped and measured 40–100 nm in diameter. Exosomes are naturally secreted and well tolerated by the body. They are also safely stored and provide many therapeutic applications with avoiding the

7. Conclusion

Abbreviations

ESCs embryonic stem cells

FSCs fetal stem cells

iPS induced pluripotent stem cells

MSCs mesenchymal/stromal cells

SEM scanning electron microscope

Rab Ras-related proteins in brain

GTP guanosine triphosphate

Amany A. Moneim Solaiman

University, Alexandria, Egypt

Medicine. 2008;2(4):169-183

TEM transmission electron microscope

SNARE soluble N-ethylmaleimide-sensitive factor receptor

Address all correspondence to: amanysolaiman@gmail.com

Department of Medical Histology and Cell Biology, Faculty of Medicine, Alexandria

[1] Bajada S, Mazakova I, Richardson JB, Ashammakhi N. Updates on stem cells and their applications in regenerative medicine. Journal of Tissue Engineering and Regenerative

CD cluster of differentiation

MMP metalloproteinases

P passage

Author details

References

promising candidate for drug delivery vehicle.

The MSCs maintained their undifferentiated histological structure till passage 9 for further tissue engineering. A detailed histological examination using the light and the electron microscopes is essential to understand the function of MSCs. In addition, exosomes represent a

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Figure 11. Transmission electron micrograph of cultured human bone marrow derived stromal cells. The cell surface of shows an open fibripositors (short arrow) with large amounts of secretory product (S) is observed. Note the euchromatic nucleus (N) [38]. Scale bar 0.5 μm.

risk of immunological rejection and malignant transformation [65]. Therefore, the use of MSCs to produce exosomes for drug delivery is the subject of the day [66]. Recently, liposomes are preferred drug delivery systems. It is a synthetic vesicle with a phospholipid membrane that has the ability to self-assemble into various sizes and shapes in an aqueous environment [67].

Another morphological feature detected is pseudopodia-like structures extending from the cell membrane (Figures 7 and 10). This might explain the capacity of the cells for migration within the receiving tissue. The main role of these structures is to transmit the produced material from one cell into another by extending the pseudopodia and communicating cells with each other as well as in cell signaling [68]. Interestingly, one of the most striking features during differentiation is the observation of finger-like extensions of the plasma membrane known as fibripositors (Figure 11). These fibripositors were located at the side of the cell and protrude into the spaces between cells. These fibripositors are the site where collagen fibrils were located. It was reported that the initial stage of extracellular matrix deposition results in arrays of short collagen fibrils completely enclosed within these fibripositors. These fibrils are then subsequently deposited extracellularly [69, 70].

It was reported that fibrils leaving the fibripositors were seen to run along the external surface of the cell. Tracking of fibrils revealed that the collagen fibrils in fibripositors were shorter than those extracellularly. Thus, these data suggested that fibripositors might be a place of fibril assembly. They determined that short fibrils become longer inside closed fibripositors, then protruding fibripositors (open), often project into the matrix, releasing fibrils extracellularly where individual fibrils then coalesce into bundles. Thus, fibripositors are specialized sites not only of fibril assembly, but also share in fibril transport extracellularly [71].

Another study declared that the fibripositors are dynamic structures and their formation and stabilization depend on the actin cytoskeleton [72]. This might explain the existence of the cytoskeletal filaments in the differentiating cells [38]. Accordingly, these cytoskeletal structures might be actin filaments. It is possible that fibripositors have been involved in the alignment of extracellular collagen fibrils in a parallel arrangement [73].
