Chapter 5 - hAFS cells for liver repair - Final...

Chapter 5

HUMAN AMNIOTIC FLUID STEM CELLS AS A SOURCE OF HEPATIC AND ENDOTHELIAL PROGENITORS

FOR IN VIVO CELL THERAPY

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ABSTRACT

Human amniotic fluid has been utilized in prenatal diagnosis for over 70 years. It has proven to

be a safe, reliable, and simple screening tool for a wide variety of developmental and genetic

diseases. There is now evidence that amniotic fluid may have more utility than only as a

diagnostic tool, and it may be a source of a powerful therapy for a multitude of congenital and

adult disorders. In this chapter, we describe the development of the amniotic fluid in the human,

and the cells that are found within the amniotic fluid. We will further describe previous research

suggesting that amniotic fluid have progenitor cells, and the recent data from our lab

demonstrating a rare population of stem cells from amniotic fluid, termed amniotic fluid stem

(AFS) cells that proliferate readily in culture and differentiate into various cell types. We will

also confirm that these cells don’t form teratomas and show their potential to integrate in

regenerating tissues in vivo. It is possible that in the near future, we will see the development of

therapies utilizing progenitor cells isolated from amniotic fluid for the treatment of newborns

with congenital malformations as well as in adults using cryopreserved amniotic fluid stem cells.

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5.1 INTRODUCTION

Amniotic fluid derived stem (AFS) cells can be obtained from a small amount of an amniotic

fluid specimen during amniocentesis, a procedure that is frequently performed in many of the

pregnancies in which exists a risk of congenital abnormality of the fetus. Analysis of cell cultures

from this source, provide evidence that they may represent a new source for the isolation of cells

with the potency to differentiate into different cell types, suggesting a new source of cells for

research and regenerative medicine.

5.1.1 DEVELOPMENT AND CHARACTERIZATION OF AMNIOTIC FLUID

Amniotic Fluid in Developmental Biology

Embryonic and fetal cells derived from all three germ layers and extraembryonic tissues have

long been identified in the amniotic fluid1-3

. Regardless of the fact that the specific origins of

many subsets of these cell types still remain to be determined, their origin in the amniotic fluid is

closely tight with the developmental processes unfolding in utero at a particular gestational age.

It is not surprising that the profile of the cellular component of the amniotic fluid varies with

gestational age4.

A major milestone in early post implantation development is gastrulation. At about embryonic

day 15 (E15), gastrulation begins in the posterior region of the human embryo. Pluripotent

epiblast cells are allocated to the three primary germ layers of the embryo (ectoderm, mesoderm,

and endoderm) and germ cells, which are the progenitors of all the tissue lineages as well as the

extraembryonic mesoderm of the yolk sac, amnion, and allantois5. The latter forms the umbilical

cord as well as the mesenchymal part of the labyrinthine layer in the mature chorio-allantoic

placenta. The final positions of the fetal membranes result from the process of embryonic folding

or turning, which occurs around human E21 of gestation and ‘‘pulls’’ the amnion and yolk sac

around the embryo. The amniotic cavity pushes in also at the cranial and caudal ends of the

embryonic disc, thus increasing the degree of longitudinal folding at the head and tail folds. The

amniotic cavity also pinches the connection of the yolk sac and gut to form the narrowed

communication of the vitello-intestinal (or vitelline) duct6.

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Once formed, the amniotic sac is a tough but thin transparent pair of membranes that holds a

developing embryo (and later fetus) until shortly before birth. The inner membrane, the amnion,

contains the amniotic fluid and the fetus. The outer membrane, the chorion, contains the amnion

and is part of the placenta6, 7

. With all this developmental processes unfolding, it is not surprising

that a wide variety of different origins has been suggested for the mixture of cells within

amniotic fluid1-3, 8-11

.

Amniotic fluid Secretion and Dynamics

In humans, as mentioned above, the amniotic cavity starts to form immediately prior to

gastrulation (E8-9). Once the lining of the amnion is completed, the amniotic cavity fills with

amniotic fluid secreted by the cells in the amniotic membrane. This fluid serves as a shock

absorber for the developing embryo while prevents its desiccation. It also ensures symmetrical

structure development and growth maintaining consistent pressure and temperature. The fluid

permits freedom of fetal movement, important for musculoskeletal development and blood flow6,

7, 12.

In the first half of gestation, most of the amniotic fluid results of active sodium and chloride

transport across the amniotic membrane and fetal skin, with concomitant passive movement of

water13

. In the second half of gestation, most of the fluid comes from fetal urine. An additional

major source of amniotic fluid is secretion from the respiratory tract. Fetal swallowing and

gastrointestinal tract excretions, while not voluminous, also play a role in amniotic fluid

composition14-16

.

These fluid dynamics are responsible for the production and turnover of the amniotic fluid and

are also thought to be important in determining the cell types present in the amniotic cavity. Still,

much remains to be clarified about the ontogeny of many subsets of amniocytes at any

gestational age13, 16

.

Cell Populations in Amniotic Fluid

Amniotic fluid specimens have been long used as a source of human cells in prenatal sex

determination and diagnosis of genetic disorders. These cells also constituted early on a precious

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source of human cells for the purpose of biological investigation17-19

. Several morphologic cell

types can be found in in vitro cultures of amniotic fluid. These cells have been classified into

three generic different classes, according to their clonal growth and morphology. The most

frequent are the AF-Type of colonies which are constituted by small cells with densely staining

nuclei and only subtly distinct from “classical” fibroblasts. With the greatest in vitro growth

potential of all colony-forming cells are the F-Type colony cells that consist in fibroblast-like

cells. The other clonal cell population usually identified is the E-Type colony cells that have

large polygonal shape with smooth margins and grow in intimate contact with each other. The

morphologies found on some of these cultured cells (e.g. epithelioid, fibroblastoid, etc) also

confirms the putative origin attributed to cells in amniotic fluid as cells with origin in the fetal

amnion, skin, urinary, respiratory, and gastrointestinal tracts that can be shed into the amniotic

cavity and fluid1-3

.

Despite the fact that cell clonal populations were identified in amniotic fluid culture quite early,

it was only in 1993 that small, nucleated, round cells identified as hematopoietic progenitor cells

were found as the first reported progenitor population present in amniotic fluid20

. A study in

1996 was the first to suggest the possibility of multi-lineage potential of non-hematopoietic cells

present in the amniotic fluid, by showing myogenic differentiation of amniocytes21

.

The presence of mesenchymal cells in the amniotic fluid has also been proposed for decades22, 23

.

Nonetheless, the differentiation potential of mesenchymal amniocytes started to be unveiled only

very recently24-27

. In 2001, Kaviani et al reported that only 2 milliliters of amniotic fluid can give

about 20,000 cells, 80 % of which are viable28, 29

. Further, Tsai et al reported that amniotic fluid

cells takes 20 to 24 hours to double the number of cells collected, which is faster than umbilical

cord stem cells (28 to 30 hours) and bone marrow stem cells (over 30 hours)30

. The proliferation

rate of the cells is a very important feature for using the cells for urgent medical conditions. The

isolation success rate for amniotic fluid cells is close to 100 percent, whereas, scientists have

only been able to isolate and differentiate on average just 30 percent of mesenchymal stem cells

(MSC) extracted from a child’s umbilical cord shortly after birth30, 31

. Furthermore, extracting the

cells from one’s own amniotic fluid bypasses the problems associated with a technique called

donor-recipient HLA matching, which involves transplanting cells32

.

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Similarly, the presence of embryonic stem cells in the amniotic fluid was suggested only in the

last couple of years32-34

. Prusa et al. demonstrated Oct-4 expressing cells and neurogenic cells in

human amniotic fluid35-37

. In 2001, our lab initially reported that we could isolate a

subpopulation in human amniotic fluid that could differentiate into multiple lineages38

. In

January 2007, we reported the isolation of clonal lines of amniotic fluid stem (hAFS) cells that

express embryonic and adult stem cell surface markers39

.

Human amniotic epithelial cells (i.e. cells isolated from the Amnion derived from term placentas,

not from the amniotic fluid) have also shown multipotent potential to differentiate to all three

germ layers - endoderm (liver, pancreas), mesoderm (cardiomyocyte), and ectoderm (neural and

glial cells) in vitro40-43

.

The identification of several stem/progenitor cell populations in amniotic fluid, suggested that

assessment of expression of specific markers is vital to fully elucidate the presence of certain

stem/progenitor cell populations and number of these cells in amniotic fluid. Flow cytometric

analysis of cells obtained from primary cultured cells without expansion of amniocentesis

specimens, revealed low cell numbers for most of the stem/progenitor cell markers analyzed45

(Table 5.1).

Table 5.1. Flow cytometric analysis of cells obtained from primary

cultured cells without expansion of amniocentesis specimens.

Values are mean ± s.e.m. for five independent amniotic fluid

specimens.

Marker Percentage Positive

CD117(c-Kit) 0.9±0.2

CD34 2.8±0.3

CD105 19.5±1.1

CD133 0.9±0.2

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This result suggested that sorting of specific stem/progenitor cell populations from amniotic fluid

could be made possible using an appropriate method and/or technology. The use of anti c-kit

isolation of AFS cells exposed a new population of clonal stem cells for use in human tissue

engineering and regenerative medicine.

5.1.2 AFS ISOLATION, CHARACTERIZATION AND DIFFERENTIATION

Isolation of AFS cells

AFS cells are separated from the amniotic liquid by centrifugation. AFS cells are allowed to

proliferate in vitro in culture medium (consisting of modified α-Modified Earls Medium, 18 %

Chang Medium B, 2 % Chang C with 15 % embryonic stem cell certified fetal bovine serum,

antibiotics and L-glutamine). In culture, the progenitor cells maintain a round shape for one

week post separation when cultured in non-treated culture dishes. In this state, they demonstrate

a very low proliferative capability. After the first week the cells begin to adhere to the plate and

change their morphology, becoming more elongated and proliferating more rapidly, with a need

for passage every 48-72 hours. No feeder layers are required either for maintenance or

expansion. Once the initial cells are established in culture, a pluripotent subpopulation of

progenitor cells can be isolated through positive selection for cells expressing the membrane

receptor c-kit. We employed immunoselection with magnetic microspheres to isolate the c-Kit –

positive population from many amniocentesis specimens.

C-kit receptor

C-kit is a protoncogene that encodes a 14.5-kD transmembrane receptor, p145 (CD117 or KIT,

or stem cell factor receptor). This receptor belongs to the class III of receptor tyrosine kinase

family45

. It was first identified as a viral transforming gene (v-kit), an oncogene derived from

the feline retrovirus HZ4-FeSV, being c-kit its normal cellular homologue. Due to its feline

origin, that this protoncogene was named as c-kit, for kitten46

. The ligand for this receptor is the

Stem Cell Factor protein, also known as Mast Cell Growth Factor or Steel Factor47

.

KIT plays a key role during fetal development, and its expression is constitutively maintained in

hematopoietic stem cells, mast cells, intraepithelial lymphocytes, germ cells, melanocytes, and

interstitial cells of Cajal. It is associated with immature stages of hematopoiesis, melanogenesis,

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osteoclast and Langerhans cells differentiation, which lose the antigen during differentiation into

more mature cells48-51

.

Several mutations were identified in W mutant mice resulting in anemia, lack of mast cells,

pigmentation defects and infertility, what emphasizes its key role in fetal development and as a

growth factor receptor52, 53

. Altered c-kit levels also occur in a variety of malignancies and

cancers, like leukemias, lymphomas, small cell lung cancer and melanoma, just to mention a

few46, 53

.

Due to the critical role of c-kit in proliferation and survival of several stem/progenitor cell

populations, it was sought as a good strategy to isolate stem/progenitor cells from amniotic fluid

cultured cells.

AFS Cell Culture and Characterization

About 0.8 % to 1.4 % of cells present in amniotic fluid and placenta have been shown to be c-kit

positive. Of these, over 90 % expressed the transcription factor Oct4 which has been associated

with maintenance of the undifferentiated state and the pluripotency of embryonic stem cells54

.

The progenitor cells derived show a high self-renewal capacity with >300 population doublings,

far exceeding Hayflick’s limit. The doubling time of the undifferentiated cells is noted to be 36

hours with little variation with passages.

These cells have been shown to maintain a normal karyotype at late passages and have normal

G1 and G2 cell cycle checkpoints. They demonstrate telomere length conservation while in the

undifferentiated state as well as telomerase activity even in late passages55

. Analysis of surface

markers shows that progenitor cells from amniotic fluid express human embryonic stage specific

marker SSEA4, and the stem cell marker OCT4, and did not express SSEA1, SSEA3, CD4,

CD8, CD34, CD133, C-MET, ABCG2, NCAM, BMP4, TRA1-60, and TRA1-81. These

characteristics demonstrate that AFS cells express some of some key markers of embryonic stem

cell phenotype, but not the full complement of markers expressed by embryonic stem cells. This

may indicate that the amniotic cells are not quite as primitive as embryonic cells, yet are more

primitive than most adult stem cells. Importantly, AFS cells do not form teratomas in vivo when

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implanted in immunodeficient mice. Lastly, cells, when expanded from a single cell, maintained

similar properties in growth and potential as the original mixed population of the progenitor

cells.

AFS Cell Differentiation

AFS cells have been shown to be pluripotent and differentiate into osteogenic, adipogenic,

myogenic, neurogenic, endothelial, and hepatic phenotypes in vitro. Each differentiation has

been performed through proof of phenotypic and biochemical changes consistent with the

differentiated tissue type of interest.

Adipocytes

To promote adipogenic differentiation, AFS cells can be induced in dexamethasone, 3-isobutyl-

1-methylxanthine, insulin, and indomethacin. The cells cultured with adipogenic supplements

change their morphology from elongated to round within 8 days. This coincides with the

accumulation of intracellular droplets. After 16 days in culture, more than 95 % of the cells have

their cytoplasm filled with lipid-rich vacuoles. Adipogenic differentiation also demonstrates the

expression of peroxisome proliferation-activated receptor γ2 (pparγ2), a transcription factor that

regulates adipogenesis, and of lipoprotein lipase through RT-PCR analysis56, 57

. Expression of

these genes is noted in the cells under adipogenic conditions but not in undifferentiated cells.

Osteocytes

Osteogenic differentiation was induced in the AFS cells with use of dexamethasone, beta-

glycerophosphate, and ascorbic acid-2-phosphate58

. The cells maintained in this medium

demonstrated phenotypic changes within 4 days with a loss of spindle shape phenotype and

development of an osteoblast-like appearance with fingerlike excavations into the cytoplasm. At

16 days, the cells aggregated, showing typical lamellar bone-like structures. In terms of

functionality, these differentiated cells demonstrate a major feature of osteoblasts which is to

precipitate calcium. Differentiated osteoblasts from the AFS cells are able to produce alkaline

phosphatase (AP) and to deposit calcium, consistent with bone differentiation. The

undifferentiated cells lacked this ability. The AFS cells in osteogenic medium express specific

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genes implicated in mammalian bone development which are AP, core binding factor A1

(cbfa1), and osteocalcin in a pattern consistent with the physiological analog.

Endothelial cells

The AFS cells can be induced to form endothelial cells (EC) by culture in endothelial basal

medium on gelatin coated dishes. Full differentiation is achieved after one month in culture;

however, phenotypic changes are noticed within one week of initiation of the protocol. EC

differentiated AFS cells show expression of human-specific endothelial cell surface marker

(P1H12), factor VIII (FVIII), VEGF Receptor-2 (KDR) and CD31 that are specific for

differentiated endothelial cells. The AFS cells do not stain for endothelial specific markers. The

AFS derived endothelial cells, once differentiated, are able to grow in culture and form capillary-

like structures in vitro.

Hepatocytes

For hepatic differentiation, the AFS cells are seeded on matrigel or collagen coated dishes at

different stages and cultured in the presence of hepatocyte growth factor, insulin, oncostatin M,

dexamethasone, fibroblast growth factor 4, and monothioglycerol for 45 days59, 601

. After 7 days

of the differentiation process, cells exhibit morphological changes from an elongated to a

cobblestone appearance. The cells show positive staining for albumin at day 45 post

differentiation and also express the transcription factor HNF4α, the c-met receptor, the MDR

membrane transporter, albumin, and α-fetoprotein. Hepatic differentiation induced AFS cells

produced urea at a level of 1.21 x 103

ng urea/hour/cell compared with 5.0 x 101 ng

urea/hour/cell for the control cell populations61

.

Myocytes

Myogenic differentiation is induced in the AFS cells by culture in media containing horse serum

and chick embryo extract on a thin gel coat of matrigel62

. In order to initiate differentiation, the

cells are incubated in the presence of 5-azacytidine for 24 hours. Phenotypically, the cells are

organized in bundles which fuse to form multinucleated cells. These cells express sarcomeric

tropomyosin and desmin, both of which are not expressed in the undifferentiated cells.

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The development profile of AFS cells differentiating into myogenic lineages mirrors a

characteristic pattern of gene expression reflecting that is seen with embryonic muscle

development63, 64

. With this protocol, Myf6 is expressed at day 8 and suppressed at day 16. MyoD

expression is detectable at 8 days and suppressed at 16 days. Desmin expression is induced at 8

days and increases by 16 days65, 66

.

Neuronal

The AFS cells were induced in DMSO, butylated hydroxyanisole (BHA), and neuronal growth

factor67, 68

After 2 days the cells were returned to AFS growth medium lacking DMSO and BHA

but still containing NGF. The cells cultured in neurogenic conditions change their morphology

within the first 24 hours. Two different cell populations are apparent: morphologically large flat

cells and small bipolar cells. The bipolar cell cytoplasm retracts towards the nucleus, forming

contracted multipolar structures. Over the subsequent hours, the cells display primary and

secondary branches and cone-like terminal expansions. The induced cells show a characteristic

sequence of expression of neural-specific proteins. At an early stage the intermediate filament

protein, nestin, which is specifically expressed in neuroepithelial stem cells, is highly expressed.

The expressions of βIII-tubulin and glial fibrillary acidic protein (GFAP), markers of neuron and

glial differentiation, respectively, increases over time and seems to reach a plateau at about 6

days69

. The cells cultured under neurogenic conditions show the presence of the neurotransmitter

glutamic acid in the collected medium. Glutamic acid is usually secreted in culture by fully

differentiated neurons70

.

To induce the differentiation of AFS cells into dopaminergic cells a two-stage induction

procedure has been designed. AFS cells are first seeded on fibronectin coated plates and

incubated in DMEM/F12 medium supplemented with N2 and bFGF for 8 days. Under these

conditions over 80% of cells showed expression of nestin, a marker of neural stem cells. The

cells then were transferred to conditions biasing to production of dopaminergic neurons 71

. Under

these conditions, a fraction of these cells assumed a characteristic pyramidal morphology. Gene

expression analysis determined the expression of GIRK2 gene, a marker of dopaminergic

neurons. After induction with another neurogenic differentiation protocol using NGF, AFS cells

acquired the ability to secrete the excitatory neurotransmitter L-glutamate in response to

stimulation by potassium ions.

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5.1.3 IMPLANTATION AFS CELLS IN VIVO AND IN ANIMAL MODELS OF REGENERATION

To investigate if differentiated hAFS cells can truly contribute to the regenerative process of a

damaged organ or tissue or to new tissue growth, several animal models were used.

Although before any in vivo experiment or therapeutical application, non-tumorigenic behavior

in vivo needs to be confirmed. To investigate this, immunodeficient mice were used and cells

implanted in an immunoprivileged site, the testis, in order to avoid any residual immune

surveillance that these mice could have.

Certain cells differentiated in culture from human AFS cells are capable of integrating into

normal appearing tissue structures when transplanted in vivo, under conditions promoting

regeneration or new growth. Endothelial differentiated hAFS cells were challenged to participate

in the neovascularization of a tumour72

. Myoblast differentiated hAFS were investigated in a

skeletal muscle injury model induced by cardiotoxin I73

. Similarly, hepatocyte differentiated

hAFS were challenged to contribute to liver regeneration after partial hepatectomy74

.

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5.2 MATERIALS AND METHODS

5.2.1 Teratoma analysis

Cells of one cloned human AFS cell line (hAFS-A1 at passage 14) labelled with MMP-nlacZ,

pseudotyped with VSV-G viral vector (Harvard Gene Therapy Initiative, Harvard Medical

School, Boston, MA) and mouse embryonic stem cells (mES 129 at passage 25) were injected

into the scrotal pouch of 8-week-old male rag2-/-

C57BL/6 mice (Taconic Inc., Hudson, NY,

USA). All animal care and experimental procedures were done in full compliance with the Wake

Forest University Institutional Animal Care and Use Committee, as well as all State and Federal

Guidelines Each mouse was injected with 5x106 cells of each cell line using 0.9% sodium

chloride solution (Abbott Laboratories, North Chicago, IL, USA) as vehicle. One to three months

after injection, dependent on tumour appearance, the mice were sacrificed and the scrotum

removed. Testis and tumours (where present) were fixed for 2h at 4ºC with 2 %

paraformaldehyde (Polysciences Inc., Warrington, PA, USA), 0,2 % glutaraldehyde, 0,01 %

sodium deoxycholate and 0,02 % NP-40 (Sigma-Aldrich, St. Louis, MO, USA) in PBS 1x

(Gibco-Invitrogen, Carlsbad, CA, USA). After fixation, tissues were developed for 24 h at 30 ºC

in solution with 0,1 % X-Gal (Roche Applied Sciences, Indianapolis, IN, USA) 2mM

magnesium chloride, 0,02 % NP-40, 0,01% sodium deoxycholate, 5 mM potassium ferricyanide

and 5 mM potassium ferrocyanide (Sigma-Aldrich, St. Louis, MO, USA).

After X-Gal solution development, tissue was processed in a Shandon Citadel 1000 Tissue

Processor (Thermo Electron Corporation, Waltham, MA, USA) and paraffin embedded for

sectioning. Histological examination was done using hematoxylin and eosin statining.

5.2.2 Myoblast Implantation

Cloned hAFS-A1 cells were cultured in induction medium promoting myogenic differentiation

(as described previously) and 5x105 cells previously labeled with Ad6-nlacZ vector, encoding β-

galactosidase (β-Gal) modified with a nuclear localization signal (Harvard Human Gene Therapy

Initiative, Harvard Medical School, Boston, MA) were injected using Matrigel HC (BD

Biosciences, San Jose, CA, USA) as a vehicle in male nu/nu (nude) mice (Charles River

Laboratories Inc, Wilmington, MA) 8 weeks old and 24h after injection with 30 µl of

Cardiotoxin I solution (Sigma-Aldrich, St. Louis, MO, USA) at 10-5

M in deionized water on

both hindlimbs posterior tibialis skeletal muscle. Right hindlimb muscle was used as control.

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Before cell injection, both hindlimbs were γ-irradiated with 25 gray by a JL Shepherd Mark II

Cesium-137 Irradiator. 100 µl of 0,9 % sodium chloride solution (Abbott Laboratories, North

Chicago, IL, USA) were injected in the right hindlimb muscle as control.

Samples were collected for histological analysis after 1 (n=3) and 3 weeks (n=3).

The dissected posterior tibialis skeletal muscles (hAFS injected and control) were developed in

X-Gal solution (as described above). After development, tissue was processed and sectioned.

Sections were stained with hematoxylin and eosin and immunostained with mouse anti-

sarcomeric tropomyosin (Sigma-Aldrich, St. Louis, MO, USA).

5.2.3 Endothelial Cell Implantation

Cloned hAFS-A1 cells were cultured in induction medium promoting endothelial differentiation,

and then 5x105 cells were injected together with 2x10

6 cells of human pancreatic carcinoma cells

Hs-776T (HS-VF), engineered to over-express VEGF, into immune deficient nu/nu (nude) mice

(Charles River Laboratories Inc, Wilmington, MA). The human cells were marked with the

nlacZ gene with a nuclear localization β-galactosidase, introduced via a defective adenoviral

vector (Harvard Human Gene Therapy Initiative, Harvard Medical School, Boston, MA), to

allow their detection using a chromogenic β-galactosidase (β-Gal) substrate (X-gal).

After 1 and 4 weeks tumours were collected for histological analysis. All samples were

developed with X-Gal solution, before tissue processing and sectioning as described above.

Sections were stained with hematoxylin and eosin. To detect mouse blood vessels, sections were

immunostained with rat monoclonal anti-mouse CD31 antibody (BD Pharmingen, Franklin

Lakes, NJ, USA).

5.2.4 Hepatocyte Implantation

Cloned hAFS-A1 cells were cultured in induction medium promoting hepatic differentiation (as

desbribed previously). Immune deficient nu/nu (nude) mice (Charles River Laboratories Inc,

Wilmington, MA) were submitted to a partial hepatectomy (70 %) before injection of 5x105 cells

previously marked with the nlacZ gene with a nuclear localization β-galactosidase, introduced

via a defective adenoviral vector (Harvard Human Gene Therapy Initiative, Harvard Medical

School, Boston, MA). These cells were injected in the parenchyma of the remaining liver lobe

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(right lobe) using 0.9 % sodium chloride solution (Abbott Laboratories, North Chicago, IL,

USA) as vehicle.

Samples were collected for histological analysis after 1 and 3 weeks. All samples were

developed with X-Gal solution, before tissue processing and sectioning as described above.

Sections were stained with hematoxylin and eosin and immunostained with mouse monoclonal

anti-albumin antibody (Sigma-Aldrich, St. Louis, MO, USA).

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5.3 RESULTS

Tumor formation was observed in the scrotal pouch of mice implanted with mES 129 cells as

early as 4 weeks. As expected, all mice injected with mES 129 generated massive tumors. After

histological examination, several differentiated structures/tissues were observed within the

tumors (Figure 5.1A and 5.1B), undeniably confirming their embryonic origin and their

classification as teratomas. The mice injected with hAFS, didn’t develop any tumors within the 3

month range of the study. No β-galactosidase positive cells could be observed in the histological

sections of the testis and surrounding tissues (Figure 5.1C). Nonetheless, some scattered small

vesicular X-Gal (blue) staining could be observed in some areas of the epididymis (Figure 5.1D).

This blue staining was not nuclear localized, what indicates some sort of staining artifact. This

experiment confirms the non-tumorigenic nature of hAFS cells that even in immunodeficient

mice in immunoprivileged sites don’t generate tumors.

Figure 5.1 | Histological analysis of teratomas and testis of mice injected with mES and hAFS.

Teratomas were found in mice injected with mES cells and were analyzed by histological

examination and stained with hematoxylin and eosin, showing a stratified epithelium tubular

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structure (A) and a cell mass of chondroid tissue (B). Mice injected with hAFS didn’t form any

tumors and histological analysis of testis, revealed normal tissue architecture without stem cell

incorporation (C). In some areas of the epididymis, non specific scattered X-Gal staining could

be observed, probably due to a staining artifact.

When hAFS are differentiated into myoblasts, β-Gal positive myofibers could be clearly

observed in histological sections of the posterior tibialis skeletal muscle damaged with

cardiotoxin I (Figure 5.2A) one week after cell implantation. Immunohistochemical staining with

antibody anti-sarcomeric tropomyosin confirmed the skeletal muscle identity of these labeled

myofibers (Figure 5.2B). The integration of hAFS in the damaged skeletal muscle of this animal

model shows that differentiated hAFS can identify the microenvironmental cues produced in

regenerating muscle and contribute to the repair of the injured tissue. The exact

mechanism/biology used by these cells in this regenerative process remains to be elucidated.

Figure 5.2 | Histological analysis of injured skeletal muscle and VEGF secreting tumor cells

injected with hAFS cells differentiated into myoblasts and endothelial cells, respectively. After

cardiotoxin I injection, the hAFS cells injected into mice skeletal muscle incorporated in the

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damaged myofibers area. β-gal positive myocytes can be clearly observed in a hematoxylin and

eosin staining (A). These blue myofibers express also sarcomeric tropomyosin, confirming their

skeletal muscle identity (B). In the retrieved tumors, it could be observed some β-gal positive

cells (C). After CD31 staining to identify vascular structures, it could be observed blue staining

cells aligned with the CD31 expressing cells, indication of possible contribution of hAFS cells

into these newly formed vascular structures (D).

The implanted pancreatic cancer cells developed a detectable tumour as early as 2 weeks after

injection and up to 4 weeks. Blood vessels growing in the tumour were visualized by

immunohistochemical staining with an antibody to mouse CD31. Staining for β-Gal (blue)

shows that human endothelial cells derived from hAFS cells, co-localize with these vascular

structures. This provides some evidence that hAFS cells have the potential to integrate into these

new blood vessels (Figure 5.2D) and contribute to tumour angiogenesis.

One week after hAFS cell injection into a regenerating liver, several extensive clusters of β-Gal

positive cells could be observed throughout the whole liver (Figure 5.3A and 5.3B).

Immunohistochemical staining for albumin showed intense expression within one of the clusters

of β-Gal positive cells (data not shown). Although cell nuclei imaging with PI was quite difficult

in these cell clusters, due to the intensity and darkness of the blue staining. Nevertheless, this

experiment showed that hAFS differentiated into hepatic lineage engrafted in regenerating

hepatic tissue after partial hepatectomy. However, due to the substantially different mechanisms

of hepatic tissue repair, other injury models should be used to clarify and expand the potential of

hAFS in liver disease/regeneration75

.

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Figure 5.3 | Histological analysis of regenerating mouse liver after hAFS cell injection. One

week after partial hepatectomy and hAFS cell injection, extensive clusters of β-Gal positive cells

could be observed in eosin (A) and hematoxylin and eosin stainings (B).

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5.4 DISCUSSION

The use of embryonic stem cells has been an intense debate with ethical controversies. Stem

cells in amniotic fluid may represent an attractive alternative to embryonic and adult stem cells

because of their apparent advantages of accessibility and multipotentiality over embryonic and

adult stem cells, respectively.

There are many clinical situations that one could predict amniotic fluid stem cells be used for.

For example, that one could foresee amniotic fluid stem cells being used to repair congenital

anomalies. The cells could be used for surgical reconstruction of birth defects in the neonatal

period76

. A small amount of amniotic fluid may be sufficient to yield enough cells to prepare and

engineered tissue construct for implantation ready upon birth77, 78

. An example is a recent study

showing experimental diaphragmatic hernia repair with autologous tendon engineered from

mesenchymal amniocytes79

.

The in vitro and in vivo experiments completed so far provide solid evidence of the safety and

regenerative capability of these stem cells80

, although further in vivo studies need to be

performed to expand the potential of hAFS cells in human medicine. In the particular case of the

liver, combination of hAFS derived endothelial and hepatic cells constitute a new alternative

source for liver cell therapies and liver tissue engineering. This is further emphasized by the fact

that the most important factor affecting clinical hepatocyte transplantation is a lack of donor

availability81

. Also, clinical evidence shows that adult hepatocyte transplantation benefit is

transient in its therapeutic effects, due to the limited capacity of hepatocyte proliferation, reduced

cell engraftment after transplantation and immune surveillance82

. Hepatocyte progenitor cells

constitute potentially a better approach for transplantation. Considering the properties of hAFS,

differentiation of these cells in hepatic progenitor cells is of capital importance, potentially

increasing cell availability and immune compatibility. We expect that in the near future, amniotic

fluid stem cells will become a relevant, vital tool in stem cell research, tissue engineering, and

regenerative medicine.

108

5.5 CONCLUSION

AFS cells isolated from amniotic fluid are an exciting contribution to the field of stem cell

biology and regenerative medicine. The discovery of these cells has been recent, and a

considerable amount of work remains to be done on the characterization and use of these cells in

vivo. In the future, banking of these stem cells could provide a convenient source for autologous

therapy by matching of histocompatible donor cells with recipients. AFS cells could represent an

attractive and abundant, noncontroversial source of stem cells for regenerative medicine.

109

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