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Amniotic fluid – a source for clinical therapeutics in the newborn?
Å. EKBLAD1,2, H. QIAN2,3, M. WESTGREN1, K. LE BLANC2,4 , M. FOSSUM 5,6 AND C. GÖ THERSTRÖ M1,2*
1
Department of Clinical Science, Intervention and Technology, Division of Obstetrics and
Gynecology, Karolinska Institutet, 14186 Stockholm, Sweden. 2
Center for Hematology and Regenerative Medicine, Karolinska Institutet, 14186 Stockholm,
Sweden. 3
Department of Medicine, Karolinska Institutet, 14186 Stockholm, Sweden.
4
Hematology Center, Karolinska University Hospital, 14186 Stockholm, Sweden.
5
Department of Women’s and Children’s Health, Center for Molecular Medicine, Karolinska
Institutet, 17176 Stockholm, Sweden. 6
Department of Pediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University
Hospital, 17176 Stockholm, Sweden.
Running title: Human amniotic fluid-derived cells.
Corresponding author: Cecilia Götherström, PhD Department of Clinical Sciences, Intervention and Technology Division of Obstetrics and Gynecology, K57 Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden 1 (25)
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2 Phone: +46 (0) 585 811 61, Fax: +46 (0) 585 811 64 [email protected]
AUTHOR INFORMATION Åsa Ekblad +46 (0)73 941 51 32 [email protected] Hong Qian Office: +46 (0)8 585 836 23 Fax: +46 (0)8 585 836 05
[email protected] Magnus Westgren +46 (0) 585 816 27 [email protected] Katarina Le Blanc +46 (0) 585 813 61 [email protected] Magdalena Fossum +46 (0) 517 783 10 [email protected]
Statement: This work has never been presented at conferences or submitted to any journal before.
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ABBREVIATIONS AF
Amniotic fluid
AFC
Amniotic fluid cells
CPM
Counts per minute
CFU-F
Colony forming unit-fibroblast
Ci
Curie
DMEM
Dulbecco´s modified Eagles medium
Fibr
Fibroblast
Gy
Gray
MEMα
Minimum Essential medium alpha
MSC
Mesenchymal stem cell
P
Passage
PBL
Peripheral blood lymphocytes
PD
Population doublings
PDT
Population doubling time
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ABSTRACT Congenital malformations are the leading cause of deaths during the neonatal period. Infants with prenatally diagnosed soft tissue defects may benefit from readily available autologous tissue for surgical implantation perinatally. In this study we investigated the cell content of amniotic fluid (AF) and its suitability for isolation and expansion of autologous cells for clinical use. Second trimester AF was obtained at routine amniocentesis at mean gestational week+day 16+2 (n= 54). To investigate the cell content, freshly harvested AF cells (AFC) were analyzed for different cell lineages by flow cytometry. To evaluate the isolation and expansion potential of AFC, isolation by plastic adherence was evaluated with three cell culture media and positive selection of CD117, CD133, CD271 and fibroblast cells were evaluated with Minimum Essential medium alpha (MEMα) culture media. Both the positive (+) and the negative (-) fractions were analyzed. Surface expression, senescence, immunogenicity, colony forming, proliferation and differentiation capacity was examined. In the non-hematopoietic, non-endothelial fraction of freshly harvested AF (n=17), 0.13 % cells were CD73+/CD117+ and 0.12 % CD73+/CD271+. AF displayed large donor variability with varying isolation and expansion success (n=30). The proliferative, differentiative, colony forming capacity and time before senescence also showed high variance. AFC had a preference for osteogenic rather than adipogenic lineages. Most culture-expanded AFC expressed mesenchymal but not hematopoietic surface epitopes. AFC expanded in DMEM showed higher immunogenic characteristics. This study shows that AF is a heterogeneous cell source, with high donor variation. Therefore it may not be the best source for autologous cell therapy.
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INTRODUCTION Approximately 2-3 % of all newborns are affected of major congenital malformations [1]. These defects are responsible for more than a third of all pediatric hospital admissions and for up to 50 % of the total cost of pediatric hospital treatment [2]. The defects are also the leading cause of deaths during the neonatal period [3]. Many pregnant women and their partners facing a prenatal diagnosis of congenital malformation in their fetus can choose between termination of the pregnancy or to deliver an affected child. The postnatal treatment of many congenital malformations involves surgical interventions, but in some cases the treatment is complicated by insufficient availability of suitable tissues or organs. The use of prosthetic materials is associated with risks that involve rejection, dislodgement, infections and lack of growth potential [4-7]. The progress in neonatal therapy makes autologous tissue engineering a rational alternative to current treatments. Candidates are malformations with severe soft tissue defects in the abdominal wall, skin and/or muscle such as omphalocele, gastroschisis, myelomeningocele, congenital diaphragmatic hernia, bladder exstrophy and cloacal exstrophy defects. It would be beneficial if the cells used for autologous tissue engineering could be harvested during the pregnancy, allowing generation of an implant to be used shortly after birth. The cell source should also be easy accessible and not impose any considerable risk for the mother or the fetus. Amniotic fluid (AF) surrounds the developing fetus within the amniotic cavity, giving the fetus protection against mechanical stress and supports the development of the fetal lungs and gut. AF is a clear fluid, primarily composed of water and electrolytes, but also includes nutrients and 5 (25)
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6 growth factors facilitating fetal growth [8]. AF also contains cells (AFC) and these cells have been used for prenatal diagnosis for over 60 years [9]. During the last decade there have been several reports on AF-derived progenitor and stem cells [10-15] and their potential for tissue engineering [16-18]. Human heart valves have been produced by seeding AFC onto a scaffold [19] and ovine autologous vascular grafts have been produced using AFC [20], suggesting that AF is a promising source for neonatal cell therapy. In cases where a severe malformation has been found with prenatal imaging or in pathological prenatal nuchal translucency screening tests, pregnant women typically are offered an amniocentesis to exclude chromosomal abnormalities, a minimally invasive procedure routinely performed in hospitals with a low risk of miscarriage (160 samples [25]. The isolation success was low (40-80 %) and they could not maintain many cultures for more than 2 passages.
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19 Studies have shown that cells from AF harvested before gestational week 20 proliferate more and display more cumulative population doublings than samples collected after week 20 [18,25]. All our samples were collected before gestational week 20, but still AF appears to be a source difficult to predict and to standardize for the purpose of autologous tissue engineering.
In freshly harvested AF we identified distinct cell populations by flow cytometry. The largest population consisted of hematopoietic cells. The different populations of non-hematopoietic, non-endothelial cells were less than 1 %, results that we confirmed with immunocytochemistry. This shows that stromal cells are rare in second trimester AF, which is in line with other reported results [12,26].
Based on the data from freshly harvested AF we derived and expanded cells from the AF by plastic adhesion and direct cell isolation using four antigens (CD117, CD133, CD271 and fibroblasts). CD117 and CD133 as well as plastic adhesion has successfully been used for isolation of cells from AF and CD271 for isolation of mesenchymal stem cells (MSC) from bone marrow [12,19,23]. We included both the negative and positive cell fractions in the study and we could detect trends in differences between these cell fractions showing that the selections were specific. For example, the total mean number of passages achieved before senescence was higher in CD133- cells (hematopoietic/endothelial cell marker [19]) and CD271+ (stromal cell marker [23]) and PDT was shorter in the CD133- cells compared to their corresponding fraction, although the total number of population doubling did not differ. This was also evident in the percent senescent cells where CD133- and CD271+ cells had significantly lower amount of 19 (25)
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20 senescent cells compared to the corresponding fraction. Notable is that not all samples could be maintained in culture to passage 10. Stromal cells do not express CD133 and Schmidt et al. showed that CD133- AFC demonstrated characteristics of MSC and CD133+ AFC showed features of functional endothelial cells [19], which may explain why CD133+ cells did not proliferate under the experimental conditions in the present study.
We investigated if AFC expressed the typical surface markers of culture-expanded MSC using flow cytometry [20]. In the present study none of the samples expressed CD14, CD45, CD31, CD80 or HLA class II and all samples displayed high expression of CD44, CD73 and HLA class I and intermediate expression of CD90 except cells grown in Chang medium that expressed CD73 and CD90 at lower levels. Interestingly all cells exhibited low expression of CD105 and some samples had low expression of CD34, similar to MSC derived from adult adipose tissue [27,28]. Several studies has shown large variations in surface expression on AFC, which has been attributed to many different features like cell shape, gestational age and cell growth conditions [24,26,29,30].
The herein used differentiation assays are well established in our laboratory, and have been proven successful in many studies [31-34], but were consequently not successful with AFC. It appears that AFC, like other fetal cells [31,35,36], have a preference for the osteogenic lineage, demonstrated by the higher osteogenic compared to adipogenic differentiation in our study and also in another study [25]. Taken together this shows that AFC is a heterogeneous population of cells and cannot be considered as true MSC specifically due to their low trilineage potential and
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21 divergent surface marker expression. Although of note is that the published criterion for MSC was created for adult cells derived from bone marrow [20].
In our study cells cultured in DMEM induced a weak immune response. None of the other cell fractions did. It might be explained by the composition of the medium or by the fact that AFC cultured in DMEM had highest expression of CD34, up to 36.9 %.
For clinical purposes millions of cells in an early passage are needed. CD117+ cells gave the highest yield after five passages; they had the shortest PDT, highest number of total passages and number of population doublings. CD117+ cells also had the lowest frequency of senescent cells and produced confluent plates in the CFU-F assay, which no other cells did. It has been published that CD117+ cells derived from AF have high potential; short population doubling time (approximately 36 h), differentiate to mesenchymal lineages and express pluripotency (Sox2, Oct4 and Nanog) and germ cell markers (DAZL and STELLA) [12,37,38]. Our study shows that among the cell populations studied, CD117+ AFC display highest proliferation potential. However, of note is that not all CD117+ cultures were successful, and the variation was high.
Although amniotic cells originate from fetal tissues, none of the AF populations reached 50 population doublings, an expansion limit (the Hayflick limit) that could be regarded as optimal for healthy cells [39]. The reason for this is unknown but one cause could be the heterogeneity of the cell population leading to only a small portion of the cells getting optimal culture conditions. The cells we aim for in our studies, MSC, might be very few but these particular cells 21 (25)
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22 might have fulfilled 50 population doublings although the total cell population had not. Another reason might be that the cells in AF were old and mostly non-functional when shed off from the fetus. In order to answer these questions, further micro-molecular evaluation of cell qualities would need to be performed including studies for chromosomal structural aberrations, gene expression profiling and telomere length, before clinical application.
In conclusion, the present study shows that AF is a heterogeneous source for isolation of cells. AF may not be a suitable source for autologous clinical tissue engineering since the donor variability is high and we could not find a reliable method to produce a standardized cell product.
Acknowledgements This study was funded by the VINNOVA (2010-00501) and by and through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institutet (20120479). We thank the Center for Fetal Medicine at Karolinska University Hospital for supplying the AF samples.
Author Disclosure Statement No competing financial interests exist.
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FIGURE LEGENDS
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Figure 1. The diverse morphology in primary amniotic fluid cell cultures. (A) CD133+ cells at day 9 in P0. The arrow indicates a dense colony consisting of epithelial-like cells. (B) Cells isolated by plastic adherence in MEMα at day 11 in P0. (C) CD117+ cells at day 9 in P0. (D) CD117- cells in P10. Magnification 10x.
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Figure 2. Mean days in P0 and number of cells retrieved at the first harvest. (A) Days of AFC in passage (P) 0 before first passage. (B) Harvested cells at the first passage per ml amniotic fluid (AF). The data is normalized to the ml starting volume of AF. The line shows the median number of cells. (C) Accumulated number of cells retrieved at passage 5 (mean ± stdev). The amount is normalized to culture area and is given per cm2. Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation.
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Figure 3. The total number of population doublings and population doubling time of amniotic fluid cells. (A) Total number of population doublings (PD) achieved by amniotic fluid cells (AFC) until senescence (mean+stdev), n=1-4, depending on isolation and expansion success rate. (B) Mean population doubling time (PDT) and number of passages in culture. Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation.
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Figure 4. Frequency of senescence among amniotic fluid cells after expansion. (A) AFC were isolated and expanded until passage 6 and 10 and stained for Beta-Galactosidase (blue staining) as a sign for senescence. Percent positive cells were quantified by counting positive and negative cells in three random view fields per well. Typical picture of BetaGalactosidase positive cells from fractions of (B) CD117+ cells at P6 and (C) Fibr+ cells at P6. Magnification 10x. (-) = no cells grew to passage 10.
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Figure 5. The ability of amniotic fluid cells to form single cell-derived colonies. Triplicates of 40 cells from passage 3 were plated per 6-well plate and cultured for 14 days. The number of colonies (>50 cells/colony) was counted in each well after staining in Eosin. CD117+ cells could not be analyzed because of 100 % confluence. Cells cultured in Chang could not be maintained in culture up to passage 10. (A) Number of colony forming unit-fibroblast (CFUF)/120 seeded cells. The median number of cells displayed with a line. A typical colony of (B) Fibr- cells and (C) CD271- cells. Magnification 4x. n=1-5 depending on isolation and expansion success rate.
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Figure 6. Differentiation potential of AFC into osteogenic and adipogenic lineages in vitro. (A) The differentiation ability in all groups is summarized in the table. The frequency of samples that differentiated into the given lineages is given in percent. (B) Typical picture of osteogenic differentiation of CD117- cells and staining with Alizarin Red S for minerals (red staining). 10x magnification. (C) Typical picture of adipogenic differentiation of Fibr- cells and staining with Oil Red O for lipid vacuoles (red staining). 20x magnification. The samples were considered as positive when the majority of the cells had differentiated, confirmed by positive staining. As negative control cells were cultured with standard expansion medium before staining. Pos =
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positive fraction after cell isolation; Neg = negative fraction after cell isolation. n = number of
samples.
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Figure 7. Immunogenicity of amniotic fluid cells. Irradiated AFC were co-cultured with lymphocytes for six days to measure the immune response. The proliferation among the lymphocytes was measured by tritium incorporation in the DNA. The positive control (POS) consisted of lymphocytes co-cultured with irradiated lymphocytes pooled from six donors and was set to 100%. Lymphocytes co-cultured with irradiated lymphocytes from the same donor served as negative control (NEG). AFC alone served as background. n = 1-4 samples per isolation method, all analyzed with two different lymphocyte donors). 36 (25)
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Figure legends for supplementary figures
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Supplementary Figure 1.
Freshly harvested amniotic fluid from 2-3 donors (n=7) was pooled and stained with CD31,
CD45, CD73, CD117, CD271 and CD235 and analyzed by flow cytometry. The figure shows the
gating strategy of a representative sample.
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Supplementary Figure 2.
The expression of surface epitopes by amniotic fluid cells was analyzed by flow cytometry at
passage 3 to 5. The figure shows the histograms of a representative sample.
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40
Plastic adherence
Table I Success of amniotic fluid cell (AFC) expansion related to isolation methods and cell culture conditions. Mean volume Isolation success in Isolation method and cell culture medium n AF in ml percent (range) DMEM media: Dulbecco’s modified Eagle medium-low glucose (HyClone Laboratories), 10 % FSC (PAA Laboratories)
4
8 (7-9.5)
100
Chang media: MEMα (Invitrogen), 15 % FCS (PAA Laboratories), 18 % Chang B (Irvine Scientific), 2 % Chang C (Irvine Scientific)
4
6 (5-8)
100
MEMα media: Minimum Essential medium alpha (Invitrogen), 15 % FCS (PAA Laboratories), collagen IV (Sigma)
4
5 (3.5-5)
100
MEMα media and CD117 4 MEMα media and CD133 5 MEMα media and CD271 5 MEMα media and Fibroblast marker 3 Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation. Positive selection
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7 (7-8) 7 (6-8) 7 (6.5-8) 6 (5.5-7)
Pos 50 100 20 100
Neg 100 100 67 100
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41
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42 Table II Identification of cell populations in amniotic fluid. Cells in freshly harvested amniotic fluid (n=17) was characterized for their surface expression by flow cytometry. 2-3 samples were pooled before flow cytometry analysis. All live nucleated cells were gated and further analyzed as shown in the table and in Supplementary Figure 1. Mean frequency of cells in % ± stdev
CD31-/CD45+/CD235+ subset (46.2 ± 24.7) CD31-/CD45-/CD235- subset (28.5 ± 32.8)
CD117+/CD73+
CD117+/CD73-
CD271+/CD73+
CD271+/CD73-
8.66 ± 6.1
51.9 ± 21.1
0.44 ± 0.4
0.27 ± 0.3
0.13 ± 0.2
0.34 ± 0.7
0.12 ± 0.1
0.08 ± 0.1
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43 Table III. Phenotype of cultured amniotic fluid cells. The expression of surface epitopes in amniotic fluid cells was analyzed by flow cytometry at passage 3 to 5.
Surface expression DMEM
Chang
MEMα
n=4
n=4
n=2
CD14
0
0
0
CD45
0
0
0
0
0
0
0
0
0
0
0
CD80
0
0
0
0
0
0
0
0
0
0
0
HLA-II
0
0
0
0
0
0
0
0
0
0
0
CD31
0
0
0
0
0
0
0
0
0
0
0
CD34
+
0
0
0
0
+
0
0
+
0
0
CD44
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
CD73
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
CD90
++
+
++
++
++
+++
++
++
++
++
++
CD105
++
+
+
+
+
+
+
+
+
+
+
Surface epitope
CD117 Pos Neg n=2 n=4 0 0
CD133 Pos Neg n=2 n=2 0 0
CD271 Pos Neg n=1 n=2 0 0
Fibr Pos Neg n=2 n=3 0 0
HLA-I +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Expression is given as negative (0) = 0-5 %, low (+) = 5-20 %, moderate (++) = 20-80 % or high (+++) = > 80 %. Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation. n=number of experiments. The experiments were carried out at least three times.
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1
Amniotic fluid – a source for clinical therapeutics in the newborn?
Å. EKBLAD1,2, H. QIAN2,3, M. WESTGREN1, K. LE BLANC2,4 , M. FOSSUM 5,6 AND C. GÖ THERSTRÖ M1,2*
1
Department of Clinical Science, Intervention and Technology, Division of Obstetrics and
Gynecology, Karolinska Institutet, 14186 Stockholm, Sweden. 2
Center for Hematology and Regenerative Medicine, Karolinska Institutet, 14186 Stockholm,
Sweden. 3
Department of Medicine, Karolinska Institutet, 14186 Stockholm, Sweden.
4
Hematology Center, Karolinska University Hospital, 14186 Stockholm, Sweden.
5
Department of Women’s and Children’s Health, Center for Molecular Medicine, Karolinska
Institutet, 17176 Stockholm, Sweden. 6
Department of Pediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University
Hospital, 17176 Stockholm, Sweden.
Running title: Human amniotic fluid-derived cells.
Corresponding author: Cecilia Götherström, PhD Department of Clinical Sciences, Intervention and Technology Division of Obstetrics and Gynecology, K57 Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden 1 (25)
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2 Phone: +46 (0) 585 811 61, Fax: +46 (0) 585 811 64 [email protected]
AUTHOR INFORMATION Åsa Ekblad +46 (0)73 941 51 32 [email protected] Hong Qian Office: +46 (0)8 585 836 23 Fax: +46 (0)8 585 836 05
[email protected] Magnus Westgren +46 (0) 585 816 27 [email protected] Katarina Le Blanc +46 (0) 585 813 61 [email protected] Magdalena Fossum +46 (0) 517 783 10 [email protected]
Statement: This work has never been presented at conferences or submitted to any journal before.
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ABBREVIATIONS AF
Amniotic fluid
AFC
Amniotic fluid cells
CPM
Counts per minute
CFU-F
Colony forming unit-fibroblast
Ci
Curie
DMEM
Dulbecco´s modified Eagles medium
Fibr
Fibroblast
Gy
Gray
MEMα
Minimum Essential medium alpha
MSC
Mesenchymal stem cell
P
Passage
PBL
Peripheral blood lymphocytes
PD
Population doublings
PDT
Population doubling time
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ABSTRACT Congenital malformations are the leading cause of deaths during the neonatal period. Infants with prenatally diagnosed soft tissue defects may benefit from readily available autologous tissue for surgical implantation perinatally. In this study we investigated the cell content of amniotic fluid (AF) and its suitability for isolation and expansion of autologous cells for clinical use. Second trimester AF was obtained at routine amniocentesis at mean gestational week+day 16+2 (n= 54). To investigate the cell content, freshly harvested AF cells (AFC) were analyzed for different cell lineages by flow cytometry. To evaluate the isolation and expansion potential of AFC, isolation by plastic adherence was evaluated with three cell culture media and positive selection of CD117, CD133, CD271 and fibroblast cells were evaluated with Minimum Essential medium alpha (MEMα) culture media. Both the positive (+) and the negative (-) fractions were analyzed. Surface expression, senescence, immunogenicity, colony forming, proliferation and differentiation capacity was examined. In the non-hematopoietic, non-endothelial fraction of freshly harvested AF (n=17), 0.13 % cells were CD73+/CD117+ and 0.12 % CD73+/CD271+. AF displayed large donor variability with varying isolation and expansion success (n=30). The proliferative, differentiative, colony forming capacity and time before senescence also showed high variance. AFC had a preference for osteogenic rather than adipogenic lineages. Most culture-expanded AFC expressed mesenchymal but not hematopoietic surface epitopes. AFC expanded in DMEM showed higher immunogenic characteristics. This study shows that AF is a heterogeneous cell source, with high donor variation. Therefore it may not be the best source for autologous cell therapy.
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INTRODUCTION Approximately 2-3 % of all newborns are affected of major congenital malformations [1]. These defects are responsible for more than a third of all pediatric hospital admissions and for up to 50 % of the total cost of pediatric hospital treatment [2]. The defects are also the leading cause of deaths during the neonatal period [3]. Many pregnant women and their partners facing a prenatal diagnosis of congenital malformation in their fetus can choose between termination of the pregnancy or to deliver an affected child. The postnatal treatment of many congenital malformations involves surgical interventions, but in some cases the treatment is complicated by insufficient availability of suitable tissues or organs. The use of prosthetic materials is associated with risks that involve rejection, dislodgement, infections and lack of growth potential [4-7]. The progress in neonatal therapy makes autologous tissue engineering a rational alternative to current treatments. Candidates are malformations with severe soft tissue defects in the abdominal wall, skin and/or muscle such as omphalocele, gastroschisis, myelomeningocele, congenital diaphragmatic hernia, bladder exstrophy and cloacal exstrophy defects. It would be beneficial if the cells used for autologous tissue engineering could be harvested during the pregnancy, allowing generation of an implant to be used shortly after birth. The cell source should also be easy accessible and not impose any considerable risk for the mother or the fetus. Amniotic fluid (AF) surrounds the developing fetus within the amniotic cavity, giving the fetus protection against mechanical stress and supports the development of the fetal lungs and gut. AF is a clear fluid, primarily composed of water and electrolytes, but also includes nutrients and 5 (25)
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6 growth factors facilitating fetal growth [8]. AF also contains cells (AFC) and these cells have been used for prenatal diagnosis for over 60 years [9]. During the last decade there have been several reports on AF-derived progenitor and stem cells [10-15] and their potential for tissue engineering [16-18]. Human heart valves have been produced by seeding AFC onto a scaffold [19] and ovine autologous vascular grafts have been produced using AFC [20], suggesting that AF is a promising source for neonatal cell therapy. In cases where a severe malformation has been found with prenatal imaging or in pathological prenatal nuchal translucency screening tests, pregnant women typically are offered an amniocentesis to exclude chromosomal abnormalities, a minimally invasive procedure routinely performed in hospitals with a low risk of miscarriage (160 samples [25]. The isolation success was low (40-80 %) and they could not maintain many cultures for more than 2 passages.
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19 Studies have shown that cells from AF harvested before gestational week 20 proliferate more and display more cumulative population doublings than samples collected after week 20 [18,25]. All our samples were collected before gestational week 20, but still AF appears to be a source difficult to predict and to standardize for the purpose of autologous tissue engineering.
In freshly harvested AF we identified distinct cell populations by flow cytometry. The largest population consisted of hematopoietic cells. The different populations of non-hematopoietic, non-endothelial cells were less than 1 %, results that we confirmed with immunocytochemistry. This shows that stromal cells are rare in second trimester AF, which is in line with other reported results [12,26].
Based on the data from freshly harvested AF we derived and expanded cells from the AF by plastic adhesion and direct cell isolation using four antigens (CD117, CD133, CD271 and fibroblasts). CD117 and CD133 as well as plastic adhesion has successfully been used for isolation of cells from AF and CD271 for isolation of mesenchymal stem cells (MSC) from bone marrow [12,19,23]. We included both the negative and positive cell fractions in the study and we could detect trends in differences between these cell fractions showing that the selections were specific. For example, the total mean number of passages achieved before senescence was higher in CD133- cells (hematopoietic/endothelial cell marker [19]) and CD271+ (stromal cell marker [23]) and PDT was shorter in the CD133- cells compared to their corresponding fraction, although the total number of population doubling did not differ. This was also evident in the percent senescent cells where CD133- and CD271+ cells had significantly lower amount of 19 (25)
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20 senescent cells compared to the corresponding fraction. Notable is that not all samples could be maintained in culture to passage 10. Stromal cells do not express CD133 and Schmidt et al. showed that CD133- AFC demonstrated characteristics of MSC and CD133+ AFC showed features of functional endothelial cells [19], which may explain why CD133+ cells did not proliferate under the experimental conditions in the present study.
We investigated if AFC expressed the typical surface markers of culture-expanded MSC using flow cytometry [20]. In the present study none of the samples expressed CD14, CD45, CD31, CD80 or HLA class II and all samples displayed high expression of CD44, CD73 and HLA class I and intermediate expression of CD90 except cells grown in Chang medium that expressed CD73 and CD90 at lower levels. Interestingly all cells exhibited low expression of CD105 and some samples had low expression of CD34, similar to MSC derived from adult adipose tissue [27,28]. Several studies has shown large variations in surface expression on AFC, which has been attributed to many different features like cell shape, gestational age and cell growth conditions [24,26,29,30].
The herein used differentiation assays are well established in our laboratory, and have been proven successful in many studies [31-34], but were consequently not successful with AFC. It appears that AFC, like other fetal cells [31,35,36], have a preference for the osteogenic lineage, demonstrated by the higher osteogenic compared to adipogenic differentiation in our study and also in another study [25]. Taken together this shows that AFC is a heterogeneous population of cells and cannot be considered as true MSC specifically due to their low trilineage potential and
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21 divergent surface marker expression. Although of note is that the published criterion for MSC was created for adult cells derived from bone marrow [20].
In our study cells cultured in DMEM induced a weak immune response. None of the other cell fractions did. It might be explained by the composition of the medium or by the fact that AFC cultured in DMEM had highest expression of CD34, up to 36.9 %.
For clinical purposes millions of cells in an early passage are needed. CD117+ cells gave the highest yield after five passages; they had the shortest PDT, highest number of total passages and number of population doublings. CD117+ cells also had the lowest frequency of senescent cells and produced confluent plates in the CFU-F assay, which no other cells did. It has been published that CD117+ cells derived from AF have high potential; short population doubling time (approximately 36 h), differentiate to mesenchymal lineages and express pluripotency (Sox2, Oct4 and Nanog) and germ cell markers (DAZL and STELLA) [12,37,38]. Our study shows that among the cell populations studied, CD117+ AFC display highest proliferation potential. However, of note is that not all CD117+ cultures were successful, and the variation was high.
Although amniotic cells originate from fetal tissues, none of the AF populations reached 50 population doublings, an expansion limit (the Hayflick limit) that could be regarded as optimal for healthy cells [39]. The reason for this is unknown but one cause could be the heterogeneity of the cell population leading to only a small portion of the cells getting optimal culture conditions. The cells we aim for in our studies, MSC, might be very few but these particular cells 21 (25)
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22 might have fulfilled 50 population doublings although the total cell population had not. Another reason might be that the cells in AF were old and mostly non-functional when shed off from the fetus. In order to answer these questions, further micro-molecular evaluation of cell qualities would need to be performed including studies for chromosomal structural aberrations, gene expression profiling and telomere length, before clinical application.
In conclusion, the present study shows that AF is a heterogeneous source for isolation of cells. AF may not be a suitable source for autologous clinical tissue engineering since the donor variability is high and we could not find a reliable method to produce a standardized cell product.
Acknowledgements This study was funded by the VINNOVA (2010-00501) and by and through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institutet (20120479). We thank the Center for Fetal Medicine at Karolinska University Hospital for supplying the AF samples.
Author Disclosure Statement No competing financial interests exist.
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Pennsylvania, P University of Pittsburgh Cancer Institute, Pennsylvania 15232‐1301, P McGowan Institute of Regenerative Medicine, Pennsylvania 15219‐3110, AD Donnenberg, P University of Pittsburgh Cancer Institute, Pennsylvania 15232‐1301, P McGowan Institute of Regenerative Medicine, Pennsylvania 15219‐3110, DoM Division of Hematology/Oncology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania and CA Hillman Cancer Center Research Pavilion Suite2.42c, Pittsburgh, PA 15213‐2582, USA. (2014). Pericytes: A universal adult tissue stem cell? Cytometry Part A 81A:12-14. Lin CS, H Ning, G Lin and TF Lue. (2012). Is CD34 truly a negative marker for mesenchymal stromal cells? Cytotherapy 14:1159-63. Zhang S, H Geng, H Xie, Q Wu, X Ma, J Zhou and F Chen. (2010). The heterogeneity of cell subtypes from a primary culture of human amniotic fluid. Cell Mol Biol Lett 15:424-39. Davydova DA, EA Vorotelyak, YA Smirnova, RD Zinovieva, YA Romanov, NV Kabaeva, VV Terskikh and AV Vasiliev. (2009). Cell phenotypes in human amniotic fluid. Acta Naturae 1:98-103. Gotherstrom C, O Ringden, M Westgren, C Tammik and K Le Blanc. (2003). Immunomodulatory effects of human foetal liver-derived mesenchymal stem cells. Bone Marrow Transplant 32:265-72. Gotherstrom C, O Ringden, C Tammik, E Zetterberg, M Westgren and K Le Blanc. (2004). Immunologic properties of human fetal mesenchymal stem cells. Am J Obstet Gynecol 190:239-45. Ryden M, A Dicker, C Gotherstrom, G Astrom, C Tammik, P Arner and K Le Blanc. (2003). Functional characterization of human mesenchymal stem cell-derived adipocytes. Biochem Biophys Res Commun 311:391-7. Le Blanc K, C Gotherstrom, O Ringden, M Hassan, R McMahon, E Horwitz, G Anneren, O Axelsson, J Nunn, U Ewald, S Norden-Lindeberg, M Jansson, A Dalton, E Astrom and M Westgren. (2005). Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation 79:1607-14. Zhang P, J Baxter, K Vinod, TN Tulenko and PJ Di Muzio. (2009). Endothelial differentiation of amniotic fluid-derived stem cells: synergism of biochemical and shear force stimuli. Stem Cells Dev 18:1299-308. Guillot PV, C De Bari, F Dell'Accio, H Kurata, J Polak and NM Fisk. (2008). Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources. Differentiation 76:946-57. Bai J, Y Wang, L Liu, J Chen, W Yang, L Gao and Y Wang. (2012). Human amniotic fluidderived c-kit(+) and c-kit (-) stem cells: growth characteristics and some differentiation potential capacities comparison. Cytotechnology 64:577-89. Yu X, N Wang, R Qiang, Q Wan, M Qin, S Chen and H Wang. (2014). Human amniotic fluid stem cells possess the potential to differentiate into primordial follicle oocytes in vitro. Biol Reprod 90:73. Burnet FM. Intrinsic mutagenesis, a genetic approach to ageing. (1974). J. Wiley, New York. 25 (25)
Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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FIGURE LEGENDS
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Figure 1. The diverse morphology in primary amniotic fluid cell cultures. (A) CD133+ cells at day 9 in P0. The arrow indicates a dense colony consisting of epithelial-like cells. (B) Cells isolated by plastic adherence in MEMα at day 11 in P0. (C) CD117+ cells at day 9 in P0. (D) CD117- cells in P10. Magnification 10x.
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Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Figure 2. Mean days in P0 and number of cells retrieved at the first harvest. (A) Days of AFC in passage (P) 0 before first passage. (B) Harvested cells at the first passage per ml amniotic fluid (AF). The data is normalized to the ml starting volume of AF. The line shows the median number of cells. (C) Accumulated number of cells retrieved at passage 5 (mean ± stdev). The amount is normalized to culture area and is given per cm2. Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation.
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Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Figure 3. The total number of population doublings and population doubling time of amniotic fluid cells. (A) Total number of population doublings (PD) achieved by amniotic fluid cells (AFC) until senescence (mean+stdev), n=1-4, depending on isolation and expansion success rate. (B) Mean population doubling time (PDT) and number of passages in culture. Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation.
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Figure 4. Frequency of senescence among amniotic fluid cells after expansion. (A) AFC were isolated and expanded until passage 6 and 10 and stained for Beta-Galactosidase (blue staining) as a sign for senescence. Percent positive cells were quantified by counting positive and negative cells in three random view fields per well. Typical picture of BetaGalactosidase positive cells from fractions of (B) CD117+ cells at P6 and (C) Fibr+ cells at P6. Magnification 10x. (-) = no cells grew to passage 10.
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Figure 5. The ability of amniotic fluid cells to form single cell-derived colonies. Triplicates of 40 cells from passage 3 were plated per 6-well plate and cultured for 14 days. The number of colonies (>50 cells/colony) was counted in each well after staining in Eosin. CD117+ cells could not be analyzed because of 100 % confluence. Cells cultured in Chang could not be maintained in culture up to passage 10. (A) Number of colony forming unit-fibroblast (CFUF)/120 seeded cells. The median number of cells displayed with a line. A typical colony of (B) Fibr- cells and (C) CD271- cells. Magnification 4x. n=1-5 depending on isolation and expansion success rate.
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Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Figure 6. Differentiation potential of AFC into osteogenic and adipogenic lineages in vitro. (A) The differentiation ability in all groups is summarized in the table. The frequency of samples that differentiated into the given lineages is given in percent. (B) Typical picture of osteogenic differentiation of CD117- cells and staining with Alizarin Red S for minerals (red staining). 10x magnification. (C) Typical picture of adipogenic differentiation of Fibr- cells and staining with Oil Red O for lipid vacuoles (red staining). 20x magnification. The samples were considered as positive when the majority of the cells had differentiated, confirmed by positive staining. As negative control cells were cultured with standard expansion medium before staining. Pos =
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positive fraction after cell isolation; Neg = negative fraction after cell isolation. n = number of
samples.
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Figure 7. Immunogenicity of amniotic fluid cells. Irradiated AFC were co-cultured with lymphocytes for six days to measure the immune response. The proliferation among the lymphocytes was measured by tritium incorporation in the DNA. The positive control (POS) consisted of lymphocytes co-cultured with irradiated lymphocytes pooled from six donors and was set to 100%. Lymphocytes co-cultured with irradiated lymphocytes from the same donor served as negative control (NEG). AFC alone served as background. n = 1-4 samples per isolation method, all analyzed with two different lymphocyte donors). 36 (25)
Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 37 of 43
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Figure legends for supplementary figures
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Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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Supplementary Figure 1.
Freshly harvested amniotic fluid from 2-3 donors (n=7) was pooled and stained with CD31,
CD45, CD73, CD117, CD271 and CD235 and analyzed by flow cytometry. The figure shows the
gating strategy of a representative sample.
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Supplementary Figure 2.
The expression of surface epitopes by amniotic fluid cells was analyzed by flow cytometry at
passage 3 to 5. The figure shows the histograms of a representative sample.
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Plastic adherence
Table I Success of amniotic fluid cell (AFC) expansion related to isolation methods and cell culture conditions. Mean volume Isolation success in Isolation method and cell culture medium n AF in ml percent (range) DMEM media: Dulbecco’s modified Eagle medium-low glucose (HyClone Laboratories), 10 % FSC (PAA Laboratories)
4
8 (7-9.5)
100
Chang media: MEMα (Invitrogen), 15 % FCS (PAA Laboratories), 18 % Chang B (Irvine Scientific), 2 % Chang C (Irvine Scientific)
4
6 (5-8)
100
MEMα media: Minimum Essential medium alpha (Invitrogen), 15 % FCS (PAA Laboratories), collagen IV (Sigma)
4
5 (3.5-5)
100
MEMα media and CD117 4 MEMα media and CD133 5 MEMα media and CD271 5 MEMα media and Fibroblast marker 3 Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation. Positive selection
Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro
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7 (7-8) 7 (6-8) 7 (6.5-8) 6 (5.5-7)
Pos 50 100 20 100
Neg 100 100 67 100
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42 Table II Identification of cell populations in amniotic fluid. Cells in freshly harvested amniotic fluid (n=17) was characterized for their surface expression by flow cytometry. 2-3 samples were pooled before flow cytometry analysis. All live nucleated cells were gated and further analyzed as shown in the table and in Supplementary Figure 1. Mean frequency of cells in % ± stdev
CD31-/CD45+/CD235+ subset (46.2 ± 24.7) CD31-/CD45-/CD235- subset (28.5 ± 32.8)
CD117+/CD73+
CD117+/CD73-
CD271+/CD73+
CD271+/CD73-
8.66 ± 6.1
51.9 ± 21.1
0.44 ± 0.4
0.27 ± 0.3
0.13 ± 0.2
0.34 ± 0.7
0.12 ± 0.1
0.08 ± 0.1
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Stem Cells and Development Amniotic fluid – a source for clinical therapeutics in the newborn? (doi: 10.1089/scd.2014.0426) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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43 Table III. Phenotype of cultured amniotic fluid cells. The expression of surface epitopes in amniotic fluid cells was analyzed by flow cytometry at passage 3 to 5.
Surface expression DMEM
Chang
MEMα
n=4
n=4
n=2
CD14
0
0
0
CD45
0
0
0
0
0
0
0
0
0
0
0
CD80
0
0
0
0
0
0
0
0
0
0
0
HLA-II
0
0
0
0
0
0
0
0
0
0
0
CD31
0
0
0
0
0
0
0
0
0
0
0
CD34
+
0
0
0
0
+
0
0
+
0
0
CD44
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
CD73
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
CD90
++
+
++
++
++
+++
++
++
++
++
++
CD105
++
+
+
+
+
+
+
+
+
+
+
Surface epitope
CD117 Pos Neg n=2 n=4 0 0
CD133 Pos Neg n=2 n=2 0 0
CD271 Pos Neg n=1 n=2 0 0
Fibr Pos Neg n=2 n=3 0 0
HLA-I +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ Expression is given as negative (0) = 0-5 %, low (+) = 5-20 %, moderate (++) = 20-80 % or high (+++) = > 80 %. Pos = positive fraction after cell isolation; Neg = negative fraction after cell isolation. n=number of experiments. The experiments were carried out at least three times.
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