Pituitary stem cells: candidates and implications

Farshad Nassiri • Michael Cusimano • Jeff A. Zuccato •

Safraz Mohammed • Fabio Rotondo • Eva Horvath •

Luis V. Syro • Kalman Kovacs • Ricardo V. Lloyd

� Springer Science+Business Media New York 2013

Abstract The pituitary is the master endocrine gland of

the body. It undergoes many changes after birth, and these

changes may be mediated by the differentiation of pituitary

stem cells. Stem cells in any tissue source must display

(1) pluripotent capacity, (2) capacity for indefinite self-

renewal, and (3) a lack of specialization. Unlike neural

stem cells identified in the hippocampus and subventricular

zone, pituitary stem cells are not associated with one spe-

cific cell type. There are many major candidates that are

thought to be potential pituitary stem cell sources. This

article reviews the evidence for each of the major cell types

and discuss the implications of identifying a definitive

pituitary stem cell type.

Keywords Adenoma � Adenohypophysis � Marker �Pituitary � Stem cell � Tumour

The pituitary is an endocrine gland that rests within the sella

turcica at the base of the brain. It is considered the master

endocrine gland of the human body. It consists of two

components, the anterior pituitary (adenohypophysis) and

posterior pituitary (neurohypophysis), which regulate

numerous physiological processes such as metabolism,

reproduction, growth, and response to stress via the secretion

of its hormones. The adenohypophysis is composed of five

different hormone-secreting cells: somatotrophs which

secrete growth hormone (GH), lactotrophs which secrete

prolactin (PRL), corticotrophs which secrete adrenocorti-

cotropic hormone (ACTH), thyrotrophs which secrete thy-

roid stimulating hormone (TSH), and gonadotrophs whichs

ecrete luteinizing hormone and follicle stimulating hormone

(LH/FSH). The postnatal pituitary has a full set of terminally

differentiated hormone producing cells [1]; however, it

undergoes stages of growth that results in an increased glad

size [2]. Moreover, the pituitary is able to change its cellular

composition in response to different physiological condi-

tions, such as growth, pregnancy, and lactation. Furthermore,

the pituitary is able to regenerate after tissue injury [3]. The

mechanism of regeneration and cellular adaptation is

unknown, but is thought to be mediated either through the

differentiation of stem cells, transdifferentiation of differ-

entiated phenotypes, or mitoses of differentiated cells [4, 5].

The dogma that the adult brain was a static and quiescent

organ was disputed by the discovery of neural stem cells that

line the subventricular zone of the adult brain [6, 7]. In fact,

there is now evidence that supports the regeneration of brain

tissue via these stem cells [8]. Similarly, it is possible that

plasticity and regeneration within the pituitary may be sec-

ondary to the activation of adult pituitary stem cells. The

three fundamental characteristics of stem cells include:

(1) pluripotency, (2) a lack of specialization, and (3) an

indefinite self-renewal potential [9]. Moreover, cultured

stem cells characteristically form spheres of pluripotent and

undifferentiated cells that contain unique stem cell markers.

F. Nassiri (&) � M. Cusimano � J. A. Zuccato � S. Mohammed

Division of Neurosurgery, Department of Surgery, University

of Toronto, 30 Bond Street, Toronto, ON M5B 1W8, Canada

e-mail: farshad.nassiri@mail.utoronto.ca

F. Rotondo � E. Horvath � K. Kovacs

Division of Pathology, Department of Laboratory Medicine,

St. Michael’s Hospital, Toronto, Canada

L. V. Syro

Department of Neurosurgery, Clinical Medellin and Hospital

Pablo Tobon Uribe, Medellin, Colombia

R. V. Lloyd

Department of Pathology and Laboratory Medicine, University

of Wisconsin Hospital and Clinics, Madison, WI, USA

123

Pituitary

DOI 10.1007/s11102-013-0470-8

Populations of pituitary cells that contain stem cell

markers have previously been identified [10, 11]. More-

over, the minimal mitotic activity in pituitary tumors and

the existence of null-cell type and plurihormonal adenomas

suggest that pituitary stem cells may be a source of pitui-

tary tumorigenesis [12].

In contrast to the studies on neural stem cells, which have

clearly delineated astrocytes within the subventricular zone

and hippocampus as the definitive neural stem cells, there is

no consensus on which cells types can be considered as

definitive pituitary stem cells. Chromophobes were the first

groups of cells to be considered as potential pituitary stem

cells [5]. Chromophobes are a heterogenous group of cells

within the adenohypophysis that include, mesenchymal

cells, degranulated hormonal cells, and agranular cells

including folliculostellate, and marginal zone cells.

The first evidence of chromophobes as potential pituitary

stem cells candidates was published in 1969. In this study,

purified chromophobes from excised pituitaries were trans-

planted into the hypophysiotrophic area of the rat hypo-

thalamus. The transplanted chromophobes proliferated and

differentiated into bothacidophil and basophil cells [5].

Moreover, chromophobes were also found to differentiate

into mature acidophils and basophils in vitro [13]. However,

studies on chromophobes were unable to demonstrate that

these cells had the fundamental pluripotent characteristic of

stem cells or that the regenerated tissue was endocrinologi-

cally active. Nevertheless, the individual chromophobic cell

types are still considered to be major candidates for pituitary

stem cells. More recently, a ‘‘side population’’ of cells, and a

variety of other cells, have also been implicated as a potential

source of pituitary stem cells. In this review we outline and

discuss the evidence for the major pituitary stem cell can-

didates. We will also consider the implications for pituitary

stem cells in the treatment of adenomas.

Folliculostellate cells

Folliculostellate cells (FSCs) are stellate shaped epithelial

follicular cells with long extensions that spread and inter-

digitate between hormone secreting cells of the adeno-

hypohysis [14–16]. In humans FSCs line the lumen of large

follicles throughout the adenohypophysis whereas in rats

FSCs are generated in the early post-natal period and found

adjacent to the pituitary cleft [17–19]. FSCs have small

nuclei, numerous cytoplasmic polyribosomes, scant rough

endoplasmic reticulum and small Golgi apparatuses. The

FSCs are also immunopositive for S100 and for GFAP,

although these immunoreactivities are only temporary in

accordance with the FSC cell cycle [20–24].

The formation of follicles within the pituitary has been

noted as early as 6 weeks of gestation, and by 8–10 weeks

of gestation the morphology of these follicular cells is

almost identical to those seen in the adult pituitary [25].

The main difference between the fetal and postnatal pitu-

itary is the acinar architecture of the postnatal pituitary. It

has been shown that FSCs are formed by glandular cells

that surround foci of cell necroses thereby isolating the

necrotic debris [26]. This formation occurs in three phases.

First junctional complexes form between granulated cells.

Next, the cells undergo degranulation and dedifferentiation

of their cytoplasmic contents. Lastly, the cells are devoid of

granulates and ultrastructual similarities to granulated cells

[26]. In the latter stages the cells dismantle their endocrine

machinery and assume the nonendocrine phenotype of the

FSCs.

Numerous studies have been able to demonstrate that

FSCs may be a potential source of pituitary stem cells

[9, 27, 28]. For example, it has been shown that the number

of follicular cell clusters increases after gonadectomy,

secondary to the loss of negative feedback [29]. Moreover,

rodent pituitary grafts that were transplanted beneath the

kidney capsule regularly differentiated into striated muscle

cells that were in close association with folliculo-stellate

cells [30]. Similarly, following transplantation into the

kidney capsule, follicular cells both proliferate and gran-

ulate, and transform into immature acidophil and basophil

cells [31]. These results have also been confirmed in vitro.

Cultured Tpit/F1 cells, derived from pituitary stem cells

and biochemically similar to FSCs with the expression of

S100 and Ptx-1, were found to differentiate into myoglo-

bin-positive multinuclear tubular cells. These cells were

also positive for myogenin, a specific transcription factor

for skeletal muscle cells [32, 33].

More recently, FSCs in adenomatous and nontumorous

human pituitary glands were investigated [34]. The study

showed that endocrine cells of both tumorous and nontu-

morous cell types were capable of transforming into FSCs

while changing from an endocrine to non-endocrine

phenotype.

Stem/progenitor cells have the capability of forming

colonies in vitro [35]. Colony-forming assays in adult mice

have been used to characterize a population of putative stem/

progenitor cells located within the subluminal zone and

marginal zone of the pituitary cleft [36–38]. Of all anterior

pituitary cells, 0.2 % form colonies in vitro. These pituitary

colony-forming cells (PCFCs) are hypothesized to be a

subpopulation of pituitary folliculostellate (FS) cells based

on their expression of S100-B and GFAP, as well as their

ability to take up fluorescent didpeptide beta-Ala-Lys-N

epsilon-AMCA (AMCA-positive) [36, 39]. A small pro-

portion of these AMCA-positive FSCs have the capacity to

form colonies: 12.3 % in vitro and one-third in vivo [36, 38].

PCFCs are agranular, highly proliferate, and are capable of

dividing in [37, 38]. Interestingly, a small fraction (3.3 %) of

Pituitary

123

AMCA-positive/GH-negative cells grown in vivo differen-

tiated into GH-positive somatotrophs within 6 weeks [38].

Both somatotrophs and lactotrophs were seen at low levels

in vitro [36].

This subset of anterior pituitary FS cells expands as an

adherent colony and differentiates into multiple cell types.

However, without evidence of self-renewal and differentia-

tion into all five pituitary cell lineages, we cannot conclude

that PCFCs are true stem cells. PCFCs likely represent

committed progenitor cells with short-term proliferative

capabilities rather than pituitary stem cells. This assertion is

substantiated by the expression of progenitor cell associated

markers (ACE and stem cell antigen-1) in many PCFCs [37].

As previously mentioned, stem cell markers are fre-

quently used in stem cell studies. Nestin is an intermediate

filament protein that has been implicated in stem cells in

various tissues [40–42]. Nestin-positive cells have been

identified in the pituitary [43]. These nestin positive cells

were morphology similar to FSCs, and small subsets of these

cells were also S100 positive. The nestin positive cells did

not alter their morphological or proliferative activity after 10

passages suggesting the possibility of indefinite self-renewal

capacity. However, despite this, none of the nestin positive

cells differentiated into hormone secreting cells [43].

Few human pituitary studies demonstrating the stem cell

potential of FSCs exist. Two human sellar neoplasms with

simple cytoplasmic organization have previously been studied

[44]. Ultrastructural analysis showed that both neoplasms

contained a network of typical pituitary follicles and both

neoplasms displayed a striking similarity to fetal human

pituitary tissue of gestational ages 6 and 10–12 weeks. One of

the lesions displayed endocrine differentiation and immu-

noreactivities typical for FSCs. The origin of these two lesions

was concluded to be the FSC as a pluripotent adult stem cell

[44]. In a different study, 3 benign oncocytic neoplasms,

coined ‘‘spindle cell oncocytoma’’, showed no ultrastructural

follicular formation, however, based on immunoreactivity for

vimentin, S-100, and galectin-3, the investigators concluded

that the benign neoplasms had a FSC derivation [23].

Despite the numerous studies on FSCs, there is no con-

clusive evidence to define FSCs as true pituitary stem cells.

Morphological similarities between the FSCs and pituitary

stem cells are suggestive of their candidacy, however

definitive evidence is needed to show that these cells are

indeed able to differentiate into functional hormonal cells.

Marginal cells

The organogenesis of the pituitary is an extensive topic that

will not be covered in this review. However, it is important

to note that during embryogenesis, progenitor cells are

present in the marginal zone close to the pituitary cleft [1].

Marginal cells are located in the pituitary cleft and in the

marginal zone of the adult pituitary. These cells possess

immature structural characteristics that are very similar to

those seen in follicular cells. It should be noted that

granules have not been identified in marginal cells, and

there is a large number of free ribosomes and polyribo-

somes within the marginal cells [45, 46].

To our knowledge, there is only 1 published report that

supports the role of marginal cells as putative pituitary

stem cells. This study showed that, regardless of age, nestin

immunoreactivity was detected in cells that lined the

pituitary cleft adjacent to the marginal zone [43]. There is a

paucity of studies investigating the potential role for mar-

ginal cells as pituitary stem cells. Stem cell markers sug-

gest that marginal cells may be a source of pituitary stem

cells. However, future studies should investigate the self-

renewal capacity of marginal cells and their ability to

differentiate into hormone secreting pituitary cells.

Side population cells

The Hoescht-stained side populations of stem cells were

first discovered in 1996. This technique is based on the

assumption that stem cells extrude harmful substances.

When murine bone marrow cells incubated with Hoes-

cht33342, a DNA-binding dye, were analyzed with a

fluorescence activated cell sorter at two emission wave-

lengths, it was found that a side population of cells in the

double emission plot contained phenotypic markers of

multipotential hematopoetic stem cells [47]. This plot

showed a side population that extruded the dye, and a main

population that did not. Since its advent in 1996, this

technique has been used to identify side population of stem

cell-like cells in many tissues including the brain [48].

The side-population technique was also used to identify

potential pituitary stem cells. Using this technique it was

found that 71 % of these cells expressed Sca-1, a stem cell

marker [9]. Moreover, this pituitary side population

expressed Nestin, Bmi-1, and prominin-1, markers of neural

stem cells and hematopoetic stem cells. It is important to note

that this population did not express other markets of hema-

topoietic stem cells such as CD434 or c-kit. Oct-4 and

Nanog, which are expressed in embryonic stem cells, were

also expressed in the pituitary side population. The pituitary

side population also expressed high levels of Notch1 and

other markers that function in stem cell homeostasis and

early pituitary embryogenesis. Moreover, it was found that

the side population pituitary cells exclusively expressed

Lhx4, a transcription factor that is important in the early

embyrogenesis of the adenohypophysis. The main popula-

tion of cells did not express Lhx4, but rather expressed Lhx3,

suggesting that the side population can easily be identified as

Pituitary

123

high levels of Lhx4 likely function to maintain the undif-

ferentiated state of progenitor cells [9].

The pituitary side population of cells express many

genes that regulate stem-cell like phenotype. Nevertheless,

there is no conclusive evidence that these cells are true

stem cells as their capacity for self-renewal and pluripo-

tency has yet to be investigated. Future studies should be

performed to clearly identify these fundamental properties

and to also delineate which cells of the heterogenous side

population may be the source of pituitary stem cells.

Sox2-positive/Sox9-negative cells

Sox2 is a crucial transcription factor required for the function

of multiple stem cell populations, especially those within the

developing CNS [49, 50]. Sox2-positive and Stem cell

antingen-1 (Sca1)-positive cells present within Rathke’s

Pouch during development downregulate Sox2 and Sca1 as

they differentiate to form the adult anterior pituitary. How-

ever, a residual level (3 %) of Sox2-positive/Sca-1-positive

stem cells persist within the adult pituitary [10]. These Sox2-

positive cells line the pituitary cleft, but are also scattered

throughout the parenchyma. When cultured in vitro they

form pituispheres capable of proliferation, with 0.03–0.05 %

of cells self-renewing into secondary spheres [10]. The non-

renewing cells downregulate Sox2 and Sca1 but express

Sox9, nestin, and S100-B as they commit along particular

cell lineages [10, 51]. Committed Sox2-positive/Sox9-

positive cells differentiate into all 5 types of hormone-

producing cells as well as S100-B expressing FSCs. Spheres

were not restricted to one type of hormone-producing cell

suggesting that the progenitor cells remain multipotent [10].

The infrequent proliferation of Sox2-positive/Sox9-

positive cells, along with their S100B expression, suggests

that these cells represent transient-amplifying cells com-

mitted to a specific pituitary cell type [52]. In contrast, the

1 % of adult Sox2-positive cells that do not express Sox9 are

non-hormone producing, divide slowly, express stem cell

antigen-1, differentiate, and are able to generate secondary

pituispheres [10, 53]. Although these are characteristics of

stem cell populations, five passages are classically needed to

show that a given cell type is capable self-renewal. It will be

important for further studies to characterize the self-renewal

capacity of Sox2-positive/Sox9-negative pituicytes in order

to assess whether they are multipotent progenitors or a

population of self-renewing pituitary stem cells.

GFRa2/Prop1-positive stem (GPS) cells

Glial cell line-derived neurotrophic factor receptor alpha 2

(GFRa2) is a neurturin (NTN) receptor found expressed on

0.9 % of adult anterior pituitary cells lining the pituitary

cleft [54]. In addition to expressing many well-established

stem cells markers (Oct4, SSEA4, Sox2, and Sox9), these

cells also express Prophet of Pit1 (Prop1), a pituitary-

specific transcription factor required for pituitary devel-

opment [55]. These GFRa2-positive/Prop1-positive stem

(GPS) cells are slowly cycling cells that generate second-

ary pituispheres in vitro [56, 57]. It has been proposed that

the vimentin-positive cells surrounding the GPS cells

within the cleft serve to confine GPS cells within their

niche. However, in conditions of high NTN concentrations

outside this niche, GPS cells migrate out and differentiate

into Cdk4-positive/Oct4-negative/Prop1-negative/GFPa2-

negative transient amplifying pituicytes [56]. This expres-

sion profile is thought to represent a transition from the

differentiation-inhibiting effects of Prop1 towards the dif-

ferentiation-promoting control of Cdk4 [58]. GPS-derived

cells have the capacity to differentiate into somatotrophs,

lactotrophs, thyrotrophs, corticotrophs, gonadotrophs, and

folliculostellate cells [56].

This subpopulation of anterior pituitary cells is likely a

group of stem/progenitor cells as they are agranular,

express stem cell markers (Oct4, SSEA4, Prop1, and

Sox2), show limited self-renewal, slowly replicate, and

differentiate into all 5 types of hormone producing pituitary

cells. Interestingly, the GPS expression profile of Sox2,

Sox9, E-cadherin, and S100-B is similar the Sox2-positive/

Sox9-negative transient amplifying cells. Therefore, GPS

cells are likely a set of adult progenitor cells that proliferate

within an exclusive niche, rather than a population of true

pituitary stem cells. However, as with Sox2-positive/Sox9-

negative cells, a more extensive assessment of GPS cell

self-renewal capabilities is necessary to characterize their

phenotype.

Pituitary stem cells and pituitary adenomas

The identification of a definitive pituitary stem cell could

have major contributions to skullbase neurosurgery and

pituitary pathology. It is thought that aberrant proliferation

of pituitary stem cells occurs after an abrupt change in the

pituitary milieu leading to the formation of pituitary ade-

nomas [8, 12].

To our knowledge only 1 animal study has investigated

the role of potential stem cells in the development of pitu-

itary tumors. This study used genetic techniques to study the

role that nestin positive cells play in tumorigenesis. Mice

carrying one functional retinoblastoma allele (Rb+/-) were

crossed with nestin and GFP- positive mice [11]. The

progeny of the cross mice with predominantly melanocyte

stimulating hormone (MSH) tumors. None of the tumor cells

costained with nestin, however, nestin-positive cells that

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123

expressed Lhx3 and SOX-2 encapsulated the tumors. Based

on anatomical proximity, the authors concluded that nestin

positive cells contribute to the initiation of MSH-positive

adenomas [11].

If the hypothesis regarding pituitary tumorigenesis is valid,

the implications for pituitary adenoma treatments are significant.

Unless the aberrant pituitary stem cells are removed then an

indefinite risk exists for recurrence of the tumor even if ‘‘gross

total resection’’ of the adenoma is surgically achieved. Moreover,

if a unique cell surface marker to the aberrant stem cell is iden-

tified, then it would be possible to label the cells with an antibody

and use immunohistochemical analyses to ensure that the stem

cell is removed and to secure clear surgical margins.

In summary, the pituitary is a dynamic organ that under-

goes considerable changes in response to physiological

stimuli and pituitary microenvironment changes. These

changes are thought to be mediated by the differentiation of

pituitary stem cells. Unlike neural stem cells, pituitary stem

cells have not been clearly been delineated to a certain cell

type, and many potential candidates exist. The major candi-

dates include the chromophobes cell types, folliculostellate

cells and marginal cells,, the side population cells,

SOX2-positive/SOX9-negative cells, and GPS cells. There is

insufficient evidence to conclude that a specific cell type is

definitive of a true pituitary stem cell. There has been a con-

siderable amount of studies investigating the potential of

folliculostellate cells to act as pituitary stem cells. We believe

that the current evidence suggests that the folliculostellate

cells are the most likely cell type to represent pituitary stem

cells, however, future studies need to clearly elucidate the

pluripotent capacity of FSCs. Moreover, emerging evidence

about the candidacy of GPS cells and SOX2-positive/SOX9-

negative cells needs to be investigated further. The implica-

tions of the discovery of pituitary stem cells are considerable,

and could possibly help improve neurosurgical and medical

management of pituitary adenomas.

Acknowledgments Authors are grateful to the Jarislowsky and

Lloyd Carr-Harris Foundations for their generous support.

Conflict of interest The authors declare no conflicts of interest.

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