**4. Keratin-positive network (KPN)**

The human thymic cortex shows a fine, dense keratin network that sharply differs from that of the medulla. The medulla has a loose epithelial lattice, with small, irregular-shaped EFA. A ring-shaped, anti-cytokeratin-negative "gap" of the EFA is found between the cortex and medulla (**Figures 9** and **10**) that seems to be a unique feature for the human thymus. The chicken's thymic medullary epithelial cells form a 3D network (**Figure 11**). In some places the ring-shaped "cortico-medullary gap" has small "outpocketing" toward the medulla that shows the connection of medullary EFA with the cortico-medullary gap (CMG). The medullary and cortical epithelial cells are connected, through the CMG, with epithelial bridges of medullary-type epithelial cells. In several places the CMG is not covered by the

#### **Figure 9.**

*Thymus 1. Age 5 months. Cortical EFA is significant compared with that of the medulla. Medullary EFA is connected with the CMG (arrow).*

**13**

(**Figures 9** and **10**).

**Figure 11.**

**Figure 10.**

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses*

*Thymus 1. Age 5 months. The PS continues with the medullary EFA (arrow).*

cortex; therefore the CMG is in contact with the end of the PS. The PS reaches the CM border, widens, and become part of the medulla. Therefore, the human thymic medulla also consists of two sharply separated compartments: a keratinpositive network or lattice and an epithelium-free area. Inside the keratin-positive medullary area, few Hassall's bodies could be seen as aggregated keratin expression

*and medulla. Keratin accumulations in the medullary epithelial lattice represent Hassall's bodies.*

*Thymus 5. Age 16 months: at several places, the medullary epithelial cells, through the CMG, connect the cortex* 

In the early embryonic life of chicken, the thymic anlage appears as a primary epithelial cord, which starts to develop from the third branchial pouch of the foregut endoderm. The epithelial cord ramifies, and this ramification area develops to medullary region of the thymus [2]. Between the offshooted secondary cords,

*DOI: http://dx.doi.org/10.5772/intechopen.88588*

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses DOI: http://dx.doi.org/10.5772/intechopen.88588*

**Figure 10.** *Thymus 1. Age 5 months. The PS continues with the medullary EFA (arrow).*

#### **Figure 11.**

*Thymus*

**12**

**Figure 9.**

*connected with the CMG (arrow).*

*Thymus 1. Age 5 months. Cortical EFA is significant compared with that of the medulla. Medullary EFA is* 

positive cells in the KNA/EFA (**Figure 8**). Foxn-1 expression in the medullary KNA/ EFA is a puzzle. In early embryogenesis, Foxn-1 expresses in several mesenchymal and epithelial cells [27]; therefore one of the possibilities for solving the puzzle is that Foxn-1 is maintained in the KNA/EFA after thymus development. The other possibility is that the thymic epithelial cells induce Foxn-1 expression in mesenchymal cells of cranial neural crest origin [28]. Removal of perithymic mesenchyme at ED12 or culture of purified ED14 epithelial cells alone resulted in a threefold reduction in the bromodeoxyuridine incorporation by keratin-positive cells. Proliferation of thymic epithelial cells in the early thymus is regulated by signals from mesenchyme [29]. These mesenchymal cells produce fibroblast growth factors 7 and 10, which stimulate epithelial cell proliferation [20, 30, 31], but differentiation requires Foxn-1 [32]. The KNA/EFA [2, 9, 11] consists of mesenchyme; therefore the term

The human thymic cortex shows a fine, dense keratin network that sharply differs from that of the medulla. The medulla has a loose epithelial lattice, with small, irregular-shaped EFA. A ring-shaped, anti-cytokeratin-negative "gap" of the EFA is found between the cortex and medulla (**Figures 9** and **10**) that seems to be a unique feature for the human thymus. The chicken's thymic medullary epithelial cells form a 3D network (**Figure 11**). In some places the ring-shaped "cortico-medullary gap" has small "outpocketing" toward the medulla that shows the connection of medullary EFA with the cortico-medullary gap (CMG). The medullary and cortical epithelial cells are connected, through the CMG, with epithelial bridges of medullary-type epithelial cells. In several places the CMG is not covered by the

EFA seems to be more appropriate than the KNA that we used [2].

**4. Keratin-positive network (KPN)**

*Thymus 5. Age 16 months: at several places, the medullary epithelial cells, through the CMG, connect the cortex and medulla. Keratin accumulations in the medullary epithelial lattice represent Hassall's bodies.*

cortex; therefore the CMG is in contact with the end of the PS. The PS reaches the CM border, widens, and become part of the medulla. Therefore, the human thymic medulla also consists of two sharply separated compartments: a keratinpositive network or lattice and an epithelium-free area. Inside the keratin-positive medullary area, few Hassall's bodies could be seen as aggregated keratin expression (**Figures 9** and **10**).

In the early embryonic life of chicken, the thymic anlage appears as a primary epithelial cord, which starts to develop from the third branchial pouch of the foregut endoderm. The epithelial cord ramifies, and this ramification area develops to medullary region of the thymus [2]. Between the offshooted secondary cords,

#### **Figure 12.**

*Thymus 2. Age 18 months: the PS ends at the CMG (arrow). In the medulla, Hassall's bodies appear as solid, keratin-positive knots. Small keratin negative areas are also present in the cortex. The cortical surface of the gap is sharp.*

the mesenchyme forms the PS and capsule (**Figure 2**). The cells at the end of the secondary cords rapidly proliferate, and by day 13, the thymic tissue is histologically recognizable (**Figure 3**). In the KPN of the medulla, several epithelial cells make large surface contact, excluding lymphocytes, from Hassall's corpuscle. It is surprising that few cells of Hassall's bodies show surfactant protein B (SPB) immunoreactivity (**Figure 12**). In the medulla, scattered SPB-positive cells also occur, which like type II pneumocytes might be developed from the foregut epithelium, that is, respiratory diverticulum.

During the last century, the origin of the thymic epithelial anlage created a hot debate: namely, the epithelial rudiment develops either from the epithelium of the endodermal pouch and ectodermal cleft or solely from the endodermal pouch. At the beginning of this century, the debate seemed to be settled: in chicken-quail chimeric experiments [26] and in mouse, transplantation of the third branchial pouch epithelium under the kidney capsule [33] proved that the pouch epithelium developed to functional thymus. Furthermore, mouse chimeras with different haplotypes of class II MHC proved that only one haplotype contributed to thymic epithelial anlage [34]. However, in human thymus the presence of a sharp CMG, among the cortex and medulla, raises again the possibility of double-germ layer origin of thymic epithelial rudiment. Bargman [35], Norris [36], and von Gaudecker [9] studied the development of human thymus and came to the conclusion that the corresponding ectodermal cleft epithelium attaches and unites with the descending third pouch epithelium (**Figure 13**). Cordier and Hamount [37] compared the thymus development of NMRI and nude mice and studied that either the lack of ectodermal cleft epithelium, which surrounded the endodermal rudiment, or the absence of a secreted substance from cleft epithelium [20] resulted in the dysgenesis of nude mouse thymus. In human thymus the major EFA is represented by the "gap" (**Figures 9** and **10**).

The debate is going on, but the subject changed over the thymic epithelial stem cell, which may be also connected to the endo- and ectodermal origin. Namely, one epithelial stem cell develops to cortical and medullary progenitors (single-germ layer origin), or there are, *sui generis*, cortical and medullary epithelial stem cells (ectodermal cleft and endodermal pouch will give raise to cortical and medullary progenitors, respectively). Between cortical and medullary microenvironment, there are many differences that also may be related to the ectodermal and endodermal origin of thymic epithelial rudiment.

The double-germ layer origin of human thymic epithelial cells is supported only by reliable histological studies and functional differences: (1) cortical epithelial

**15**

(CK5+

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses*

cells contribute to T-cell maturation, and the medullary ones participate in T-cell selection. (2) CMG is present in man, but not in birds and mammals studied up to now. (3) Hassall's bodies and SPB-producing cells are found only in the medulla. (4) Thymus-blood barrier exists only in the cortex [16]. (5) In the rat and mouse

*Thymus 2. Age 18 months: anti-surfactant protein B (SPB) immunostaining recognizes Hassall's bodies and* 

above thymic dysgenesis in nude mice is related with the absence of the cleft epithelium and/or cleft-derived biological active substance, which induces branchial pouch epithelial cell proliferation [31]. In mouse and chicken, the experimental data proved that the thymic epithelial anlage develops solely from the third branchial pouch [31, 33, 38–40]. The differences in the origin of epithelial anlage between man and mouse may be traced back to evolution. In marsupials there is cervical thymus, which is purely of ectodermal origin, while the cervicothoracic thymuses have mixed ecto-endodermal [37]. Evolutionary differences in organ development between man and mouse also occur: in man the allantois has rest of urachus beyond umbilicus, while in mouse the urachus is a small rudiment, and the allantois consists of pure mesenchyme [28, 41]. The relationship between thymic epithelial cells and skin keratinocytes has been supported by serological and immunofluorescence studies in normal [29, 42] and pathological conditions [3]. These investigations provide solid evidence for cross-reactive antigens among some thymic epithelial cells, cells of

Hassall's corpuscles, and some subpopulation of skin keratinocytes [4].

and CK5+

ectodermal component to human thymic epithelial anlage.

Gupta et al. [43] studied the cytokeratin (CK5 and CK8) expression in human embryos. Before 16 weeks of gestation, the two cytokeratins are homogenously expressed in both the cortex and medulla, but after 16 weeks of gestation, the

gestational stages. This finding indirectly suggests that the cortex is the source of the epithelial progenitor cells. Norris [36] was able to show that the branchial cleft epithelium (cervical sinus) rapidly proliferates and surrounds the endodermal thymic rudiment. Thus, the presence of double-positive progenitor cells in the cortex and the rapid proliferation of cleft epithelium support the contribution of

Hassall's bodies built up from epithelial cells. The centrally locating cells of Hassall's bodies gradually keratinized, like the epidermal cells of the skin. Neutrophil granulocytes and macrophages enter the corpuscle and digest the keratinized cells [44]. Norris [36] studied the human fetal thymuses and described migration of ectodermal cells

) epithelial progenitor cells were present only in the cortex at all

K8null CD205<sup>−</sup> (in chicken the cortical thymic epithelial cells

and the medullary thymic epithelial cells are CD205<sup>−</sup>). (6) As mentioned

CD205<sup>−</sup>, and the medullary

staining, respectively. Double-positive

thymus, the cortical epithelial cells are keratin K5<sup>−</sup> K8+

*scattered positive cells in the medulla (M). The cortex (C) is free of SPB.*

epithelial cells are K5+

cortex and medulla show CK8+

and CK8+

are CD205+

**Figure 13.**

*DOI: http://dx.doi.org/10.5772/intechopen.88588*

*Compartmentalization of Human Thymic Medulla: Facts and Hypotheses DOI: http://dx.doi.org/10.5772/intechopen.88588*

#### **Figure 13.**

*Thymus*

**Figure 12.**

*gap is sharp.*

respiratory diverticulum.

"gap" (**Figures 9** and **10**).

mal origin of thymic epithelial rudiment.

the mesenchyme forms the PS and capsule (**Figure 2**). The cells at the end of the secondary cords rapidly proliferate, and by day 13, the thymic tissue is histologically recognizable (**Figure 3**). In the KPN of the medulla, several epithelial cells make large surface contact, excluding lymphocytes, from Hassall's corpuscle. It is surprising that few cells of Hassall's bodies show surfactant protein B (SPB) immunoreactivity (**Figure 12**). In the medulla, scattered SPB-positive cells also occur, which like type II pneumocytes might be developed from the foregut epithelium, that is,

*Thymus 2. Age 18 months: the PS ends at the CMG (arrow). In the medulla, Hassall's bodies appear as solid, keratin-positive knots. Small keratin negative areas are also present in the cortex. The cortical surface of the* 

During the last century, the origin of the thymic epithelial anlage created a hot debate: namely, the epithelial rudiment develops either from the epithelium of the endodermal pouch and ectodermal cleft or solely from the endodermal pouch. At the beginning of this century, the debate seemed to be settled: in chicken-quail chimeric experiments [26] and in mouse, transplantation of the third branchial pouch epithelium under the kidney capsule [33] proved that the pouch epithelium developed to functional thymus. Furthermore, mouse chimeras with different haplotypes of class II MHC proved that only one haplotype contributed to thymic epithelial anlage [34]. However, in human thymus the presence of a sharp CMG, among the cortex and medulla, raises again the possibility of double-germ layer origin of thymic epithelial rudiment. Bargman [35], Norris [36], and von Gaudecker [9] studied the development of human thymus and came to the conclusion that the corresponding ectodermal cleft epithelium attaches and unites with the descending third pouch epithelium (**Figure 13**). Cordier and Hamount [37] compared the thymus development of NMRI and nude mice and studied that either the lack of ectodermal cleft epithelium, which surrounded the endodermal rudiment, or the absence of a secreted substance from cleft epithelium [20] resulted in the dysgenesis of nude mouse thymus. In human thymus the major EFA is represented by the

The debate is going on, but the subject changed over the thymic epithelial stem cell, which may be also connected to the endo- and ectodermal origin. Namely, one epithelial stem cell develops to cortical and medullary progenitors (single-germ layer origin), or there are, *sui generis*, cortical and medullary epithelial stem cells (ectodermal cleft and endodermal pouch will give raise to cortical and medullary progenitors, respectively). Between cortical and medullary microenvironment, there are many differences that also may be related to the ectodermal and endoder-

The double-germ layer origin of human thymic epithelial cells is supported only by reliable histological studies and functional differences: (1) cortical epithelial

**14**

*Thymus 2. Age 18 months: anti-surfactant protein B (SPB) immunostaining recognizes Hassall's bodies and scattered positive cells in the medulla (M). The cortex (C) is free of SPB.*

cells contribute to T-cell maturation, and the medullary ones participate in T-cell selection. (2) CMG is present in man, but not in birds and mammals studied up to now. (3) Hassall's bodies and SPB-producing cells are found only in the medulla. (4) Thymus-blood barrier exists only in the cortex [16]. (5) In the rat and mouse thymus, the cortical epithelial cells are keratin K5<sup>−</sup> K8+ CD205<sup>−</sup>, and the medullary epithelial cells are K5+ K8null CD205<sup>−</sup> (in chicken the cortical thymic epithelial cells are CD205+ and the medullary thymic epithelial cells are CD205<sup>−</sup>). (6) As mentioned above thymic dysgenesis in nude mice is related with the absence of the cleft epithelium and/or cleft-derived biological active substance, which induces branchial pouch epithelial cell proliferation [31]. In mouse and chicken, the experimental data proved that the thymic epithelial anlage develops solely from the third branchial pouch [31, 33, 38–40]. The differences in the origin of epithelial anlage between man and mouse may be traced back to evolution. In marsupials there is cervical thymus, which is purely of ectodermal origin, while the cervicothoracic thymuses have mixed ecto-endodermal [37]. Evolutionary differences in organ development between man and mouse also occur: in man the allantois has rest of urachus beyond umbilicus, while in mouse the urachus is a small rudiment, and the allantois consists of pure mesenchyme [28, 41]. The relationship between thymic epithelial cells and skin keratinocytes has been supported by serological and immunofluorescence studies in normal [29, 42] and pathological conditions [3]. These investigations provide solid evidence for cross-reactive antigens among some thymic epithelial cells, cells of Hassall's corpuscles, and some subpopulation of skin keratinocytes [4].

Gupta et al. [43] studied the cytokeratin (CK5 and CK8) expression in human embryos. Before 16 weeks of gestation, the two cytokeratins are homogenously expressed in both the cortex and medulla, but after 16 weeks of gestation, the cortex and medulla show CK8+ and CK5+ staining, respectively. Double-positive (CK5+ and CK8+ ) epithelial progenitor cells were present only in the cortex at all gestational stages. This finding indirectly suggests that the cortex is the source of the epithelial progenitor cells. Norris [36] was able to show that the branchial cleft epithelium (cervical sinus) rapidly proliferates and surrounds the endodermal thymic rudiment. Thus, the presence of double-positive progenitor cells in the cortex and the rapid proliferation of cleft epithelium support the contribution of ectodermal component to human thymic epithelial anlage.

Hassall's bodies built up from epithelial cells. The centrally locating cells of Hassall's bodies gradually keratinized, like the epidermal cells of the skin. Neutrophil granulocytes and macrophages enter the corpuscle and digest the keratinized cells [44]. Norris [36] studied the human fetal thymuses and described migration of ectodermal cells

into the medulla. This finding may be confirmed by monoclonal antibodies (mAb(s)) (RCK 105 and RGE5) which recognize cortical epithelial cells and some medullary ones [7]. These experiments may show that cortical epithelial cells enter the medulla. Furthermore, MTS29 mAb stains isolated in medullary epithelial cells. The antigen was also present in the epidermal epithelium [4]. The marginal cells of the corpuscle are alive and perhaps temporarily capable of producing SBP (**Figure 12**) and/or other biological active substances. If we adopt the double-germ layer origin of thymic epithelial cells, then both type II pneumocytes (SPB-producing cells) and the cortical stellate cells and cells of Hassall's body are in "foreign environment" of the medulla. The surface of the cell provides important information for the neighboring cell to form tissue and organs. According to the law of thermodynamic stability, if *in vitro* two types of cells are mixed and the bond among different cells is weaker than among homotypic cells, then the cells are sorting out and the homotypic cells aggregate [45]. Possibly, this is the situation *in vivo*, in case of Hassall's body formation. Several cortical cells enter the medulla and sort out, aggregating in the form of Hassall's body. The SPB-producing type II pneumocytes have got a similar situation as cortical epithelial cells; therefore the SPB-producing cells also sorting out "join" to the Hassall's bodies, resulting in SPB-positive Hassall's corpuscles [46].
