EP2576767A1 - Process for differentiating stem cells of the amniotic membrane - Google Patents

Abstract

The present invention refers to a process for differentiating stem cells within human amniotic membrane.

Description

PROCESS FOR DIFFERENTIATING STEM CELLS OF THE AMNIOTIC MEMBRANE

The present invention refers to a process for differentiating stem cells within their natural environment.

Tissue engineering strategies usually require cell isolation and combination with a suitable biomaterial. Human amniotic membrane (AM) represents a natural two-layered sheet comprising cells with proven stem cell characteristics. Following a novel strategy, we evaluated the differentiation potential of AM in toto with its sessile stem cells as alternative to conventional approaches requiring cell isolation and combination with biomaterials.

For this, AM-biopsies were differentiated in vitro using two osteogenic media compared with control medium (CM) for 28 days. Mineralization and osteocalcin expression was demonstrated by (immuno)histochemistry. Alkaline phosphatase (AP) activity, calcium contents and mRNA expression of RUNX2, AP, osteopontin, osteocalcin, BMP-2 (bone morphogenetic protein), and BMP-4 were quantified and AM-viability was evaluated.

Under osteogenic conditions, AM-biopsies mineralized successfully and by day 28 the majority of cells expressed osteocalcin. This was confirmed by a significant rise in calcium contents (up to 27.4±6.8mg/dl d28), increased AP-activity, and induction of RUNX2, AP, BMP-2 and BMP-4 mRNA expression. Relatively high levels of viability were retained, especially in osteogenic media (up to 78.3±19.0% dl4; 62.9±22.3% d28) compared to CM (42.2±15.2% dl4; 35.1±8.6% d28).

By this novel strategy, stem cells within human AM can successfully be driven along the osteogenic pathways while residing within their natural environment. Introduction

The amniotic membrane (AM) is the innermost of the fetal membranes and is usually discarded after birth as part of the placenta. For regenerative medicine, the membrane itself as well as isolated cells thereof have shown great potential.

AM is bacteriostatic, antiangiogenic, reduces pain, suppresses inflammation, inhibits scarring, promotes wound healing and epithelialization [1-4], shows low or no immunogenicity [5, 6] and acts as an anatomical and vapor barrier [3]. Hence, it has been applied in clinical studies in wound treatment for burned skin, (decubital) ulcers, ophthalmology [7-12], as biomaterial in reconstruction of the ocular surface [13] or as an artificial vagina [14, 15], in head and neck surgery [16] as well as to prevent tissue adhesion in surgical procedures of the abdomen, head and pelvis [17-19]. Acellular AM has also been evaluated for applications such as cartilage and tendon defects [20] or peripheral nerve conduits [21]. Furthermore, since it is usually regarded as "clinical waste", it is abundantly available, and its easy procurement results in low processing costs for therapeutic application.

Morphologically, AM is a thin, highly flexible membrane composed of a human amniotic epithelial cell (hAEC) monolayer aligning on a basal membrane and the underlying stroma enclosing human amniotic mesenchymal stromal cells (hAMSC). Intriguingly, both cell types express markers of mesenchymal and embryonic stem cells [22] and can differentiate along lineages including the adipogenic, osteogenic, chondrogenic, hepatic, cardiomyogenic, and neurogenic ones [23-28]. Amniotic cells are immunomodulatory in vitro [26, 29, 30] and AM or amniotic cells have been applied in allogenic settings in clinical trials without signs of acute immune rejection [6, 31-34].

For tissue engineering applications, cells are usually applied in combination with suitable biomaterials or otherwise as intact sheets of cultured cells with the deposited extracellular matrix to circumvent carrier materials [35]. The latter technique has been applied successfully using adipose-derived stem cells to repair scarred myocardium after myocardial infarction in a rat model [36]. However, also the production of cell sheets involves isolation and cultivation of cells, which is time consuming, increases the risk for contamination with pathogens, accumulation of mutations during proliferation or a phenotypic shift during culture resulting in loss of function [28], and it might be more difficult to gain market authorization by competent authorities.

Although tissue banks usually offer non-viable, glycerol- or cryo-preserved AM for therapeutic applications, AM viability can be retained under certain in vitro culture conditions [37].

We have recognized that AM constitutes a pre-formed sheet of stem cells and were able to differentiate this biomaterial in culture into the osteogenic lineage without prior cell isolation. Thus, we present a new straightforward strategy to generate biomaterial for regenerative medicine within a short time frame and without the need to combine separate cell and biomatrix components.

Materials and Methods

Preparation and cultivation of amniotic membrane

Human placentas were collected after caesarian sections as approved by the local ethical board. Until processing, they were kept at 4°C in sterile bags (Websinger, Wolkersdorf, Austria) with Ringer lactate solution (Fresenius, Graz, Austria) containing antibiotic/antimycotic solution (penicillin G, Sandoz, Kundl, Austria; streptomycin sulphate, FATOL Riemser, Schiffweiler, Germany; amphotericin B, Bristol Meyers Squibb, Vienna, Austria). The AM was peeled off by blunt dissection, washed extensively with phosphate- buffered saline (PBS, PAA, Pasching, Austria) at 4°C and for this study dissected using 8 mm biopsy punches (Sanova, Vienna, Austria). These biopsies were transferred into 96-round bottom well plates containing 200 - 250 μΐ culture medium. Osteogenic differentiation was induced with osteogenic stimulatory kit (O it, Stem Cell Technologies, Cologne, Germany) or osteogenic medium (OM) described by Pittenger et al. [Dulbecco's modified Eagle's medium (DMEM, PAA), 10% fetal calf serum (FCS, PAA), 1% penicillin/streptomycin (PAA), 1% L-glutamine (PAA), 50 μΜ ascorbate-2-phosphate (Sigma, Vienna, Austria), 0.1 μΜ dexamethasone (Sigma), 10 nM 1 ,25-dihydroxy- vitamin D3 (Sigma), 10 mM β- glycerophosphate (Stem Cell Technologies)] [38]. As a control, biopsies were kept in control medium (CM, DMEM, 10% FCS, 1% penicillin/streptomycin, 1% L-glutamine). Medium was changed every 2-3 days.

Samples for viability analysis, alkaline phosphatase (AP) activity, calcium content, histology, immunohistochemistry and RT-PCR were taken on day 0 (dO), dl4 and d28. For calcium and AP quantification, samples were additionally taken on d7 and d21.

Viability

To visualize viability, punch-biopsies were stained with Calcein-Acetoxymethylesther (CalceinAM, Invitrogen, Lofer, Austria) and counterstained with 4',6'-Diamidino-2- phenylindol (DAPI, Abbott, Abbott Park, IL). Therefore, biopsies were incubated for 15 minutes in CM containing 5 μg/ml CalceinAM, washed with PBS and incubated for 2 minutes in PBS containing 5 ng/ml DAPI. After washing with PBS again, microscopic fluorescence pictures were taken (Axiovert 200, Zeiss, Vienna, Austria).

To quantify cell viability, an EZ4U-Nonradioactive Cell Proliferation and Cytotoxicity Assay (Biomedica, Vienna, Austria) was performed according to the manufacturer's instructions and viability calculated in % of fresh (dO) AM.

Histology

The AM specimens were fixed in 10% neutral buffered formalin for 24 hours, rinsed with PBS and stored in 70% alcohol until further processing. After dehydration in an ascending ethanol series, the AM were embedded in paraffin, sectioned (3-4 μηι) on a rotary microtome, deparaffinized in xylene, rehydrated in descending alcohol series, and stained with hematoxylin and eosin (HE). Additional paraffin sections of each specimen were stained for mineral deposits with von Kossa (v ) and Alizarin Red S (AR). Briefly, for vK staining rehydrated sections were incubated in 5% silver nitrate for 10 minutes under ultraviolet light and fixed in 5% sodium thiosulphate and slightly counterstained with 1% neutral red. For AR staining, rehydrated sections were stained with 2% AR (pH 4.1-4.3) for 30 seconds. After that, slides were rapidly dehydrated and mounted permanently.

Immunohistochemistry

All incubation steps were carried out using immunostaining chambers (Coverplate™ System, Thermo Shandon, Zug, Switzerland). Polyclonal rabbit anti-human osteocalcin (Santa Cruz Biotechnology, Santa Cruz, CA), monoclonal rabbit anti-human Ki-67 (Clone SP 6, Thermo Scientific, Fremont, CA), and polyclonal rabbit anti-human cleaved caspase-3 (Cell Signaling Technology, Danvers, MA) antibodies were used.

Sections were warmed to 56°C for 30 minutes, deparaffinized with xylene and rehydrated in a descending alcohol series. After heat induced epitope retrieval, all specimens were treated with peroxide (3% H₂0₂ in TRIS-buffered saline) for 10 minutes at room temperature to deactivate endogenous peroxidase activity. Sections were then washed with TRIS-buffered saline and incubated with the respective primary antibody (anti-osteocalcin 1 :50, anti- i-67 1 :200, anti-cleaved caspase-3 1 :100) over night at 4°C. After rinsing, sections were incubated with ImmPRESS™ anti-rabbit micropolymer (Vector laboratories, Burlinghame, CA) for 30 minutes at room temperature, washed and staining was developed by peroxidase substrate kit (NovaRED™, Vector laboratories, Burlinghame, CA). The slides were then counterstained with hematoxylin, dehydrated and mounted permanently with Roti-Histokitt II (Carl Roth, Karlsruhe, Germany). Immunohistochemical controls were performed by replacing the primary antibody with buffer.

Calcium content and alkaline phosphatase (AP) activity

For determination of the calcium content biopsies were transferred into 200 μΐ of 0.5 M hydrochloric acid (HCl, Roth, Graz, Austria) and shaken at 488 rpm for at least 3 hours at room temperature. After centrifugation, samples were evaluated with the Calcium CPC FS Kit (Diagnostic Systems International, Holzheim, Germany) according to manufacturer's instructions.

The activity of intracelluar AP was determined by freezing biopsies in PBS, permeabilizing them in 0.25% Triton-X100 (Sigma) and adding p-Nitrophenyl phosphate (p-NPP, Sigma). Samples and controls were measured at 405 nm and calculated using a p-Nitrophenol (p-NP, Sigma) standard curve.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Differentiation efficiency was evaluated by quantitative RT-PCR for specific osteogenic markers: RUNX2/CBFA1 (core binding factor alpha, Hs00231692_ml), ALPL (alkaline phosphatase, Hs00758162_ml), SPP1 (secreted phosphoprotein 1 , osteopontin, Hs00167093_ml), BGLAP (bone gamma-carboxyglutamate protein, osteocalcin, Hs01587813_gl), BMP-2 (bone morphogenetic protein-2, Hs01055564_ml) and BMP-4 (Hs00181626_ml). Therefore, biopsies were transferred into TriReagent (Sigma), frozen at - 80°C, fully thawn and dissociated by vortexing. RNA was isolated according to manufacturer's instructions. Quality and quantity of RNA were assessed by Agilent 2100 Bioanalyser (Agilent Technologies, Boblingen, Germany) using a RNA 6000 Nano Chip Kit (Agilent Technologies). Isolated RNA was processed as described by Stadler et al. using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Brunn am Gebirge, Austria) [28]. Standard curves were prepared for quantification; expression values were normalized to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) and presented as fold of induction (FOI) relative to levels in dO AM.

Calculation and Statistical analysis

For all assays 3 to 6 donors were analyzed in individual experiments. Data were tested for Gaussian distribution and analyzed using One-way ANOVA and Tukey's multiple comparison test. A p-value < 0.05 was considered as significant. Data are shown as mean±SD except for qRT-PCR data, which are shown as median with Q1/Q3 for better data presentation.

Results

Viability

To determine the effect of culture on AM viability, a quantitative assay and live/dead staining were performed. Viability of AM was significantly decreasing during in vitro culture, showing levels of 42.2±15.2%, 74.0±26.9% and 78.3±19.0% on dl4, and 35.1±8.6%, 62.9±22.3% and 55.9±12.9% on d28 for CM, OKit and OM, respectively (Fig. lA). Both osteogenic media retained viability at similar levels, whereas CM was less supportive for viability, resulting in significant reduction compared to OM on dl4. Concordantly, in live/dead staining the predominantly viable staining (green) of fresh AM was partly replaced by dead areas (blue) during culture, especially in CM (Fig.1 B).

Histology

Both media compositions for osteogenic differentiation induced mineral deposition as demonstrated by von ossa (vK) and Alizarin Red S (AR) staining of AM sections (Fig.2A). On dl4, mineral deposition was mostly associated with the epithelial layer, while on d28 also stroma cells stained positive with vK and AR. Mineralization was not distributed homogenously but rather showed patches of positive areas, both in the stromal and epithelial layer. Noteworthy, regions positively stained for vK and AR showed different cellular morphologies than negative areas as observed in HE staining (Fig.2A). In fresh (dO, Suppl. Fig.2), or CM-cultured amnion (Fig.2A), no cell-associated mineralization was detected.

To demonstrate bone-specificity of mineralization, biopsies were immunostained for the noncollagenous extracellular matrix protein osteocalcin (Fig.2A). Nearly all cells - epithelial and stromal - stained positive under osteogenic conditions. Mineralized regions (vK and AR positive) revealed intense osteocalcin staining. Non-mineralized epithelial areas were mostly characterized by nuclei situated at the membrane surface and enlarged cytoplasma, staining red with eosin (d28, osteogenic media; Fig.2A). Although not mineralized, also these epithelial cells homogenously expressed osteocalcin.

To determine the effect of culture and differentiation on viability, we further stained consecutive sections for proliferation ( i-67) and apoptosis (cleaved caspase-3). Culture in CM induced apoptosis in AM biopsies on day 14, which further increased on day 28 (Fig.2B). In contrast, hardly any apoptotic cells were detected in both osteogenic media on day 14. On day 28, caspase-3 activation was observed in distinct, mineralized areas (vK positive) under osteogenic conditions (Fig.2B). Low numbers of proliferative cells were revealed in CM on day 14 of culture but also to some extent in OM on day 14 and 28 (Fig.2B).

Calcium content and AP activity

Histological findings were confirmed by quantitative assays associated with differentiation (calcium content, AP activity). After d21 of culture, calcium contents increased significantly under osteogenic conditions (Fig.3A). Calcium contents were similar in both osteogenic media on d7 (1.6±1.2 mg/dl in OKit; 1.0±1.1 mg/dl in OM), dl4 (6.4±5.2 mg/dl in O it; 4.6±2.5 mg/dl in OM) and d21 (10.7±6.2 mg/dl in OKit; 14.3±6.2 mg/dl in OM), but increased significantly in OM (1.9-fold; 27.4±6.8 mg/dl) compared to OKit on d28. From dl4 on, also AP activity was slightly augmented in osteogenic media compared to controls, rising 1.6-fold and 2.7-fold on dl4 and d28 in OKit and 2.1-fold and 2.3-fold on dl4 and d28 in OM (Fig.3B).

Quantitative RT-PCR

Using quantitative RT-PCR, mRNA expression of selected genes involved in osteogenesis was determined on dl4 and d28. Both osteogenic media induced RUNX2 (mean 1.6±0.9 OKit; 2.0±1.1 OM), ALPL (mean 2.6±1.8 OKit; 1.6±0.9 OM), BMP-2 (mean 8.1 ±8.7 OKit; 12.8±10.1 OM) and BMP-4 (mean 5.814.7 OKit; 2.411.6 OM) expression compared to dO (Fig.4). For mRNA expression data, high donor variability was found, especially for BMP-2 and BMP-4. At mRNA level, induction of the extracellular matrix protein osteocalcin (BGLAP) was not detectable under osteogenic conditions. Also osteopontin (SPP1) expression was not upregulated and especially late during culture even downregulation of osteopontin (0.3-x on d28 in OM) was observed. Noteworthy, fresh AM already expressed osteopontin mRNA levels around 70.000-fold compared to the housekeeping gene.

Discussion

In the present proof of principle study, we have shown that viable AM in toto - with its sessile stem cell components - possesses potential as grafting material for bone regeneration. Under osteogenic conditions in vitro, the viable biomaterial became mineralized and expressed markers of early and late osteoblastic differentiation, including alkaline phosphatase, RUNX2, BMP-2, BMP-4 and osteocalcin.

Interestingly, osteogenic medium appears to support not only differentiation but also cell survival, resulting in higher viability than under control conditions. Loss of viability in control cultures may be due to increasing levels of apoptosis, as demonstrated by positive staining for active caspase-3 on day 14 and 28. Under osteogenic conditions, apoptosis was mostly limited to small, extensively calcified areas at the end of culture. Additives in osteogenic medium may rescue cells from apoptosis, and allow for subsequent differentiation. Both osteogenic media contain ascorbate, which has proven anti-oxidative and anti-apoptotic effects in vitro [39, 40] and in vivo, preventing apoptosis of endothelial cells in patients with congestive heart failure [41]. Further, ascorbate has been associated with anti-aging effects resulting in increased reprogramming efficiency for generation of human iPSC [42].

While overall viability was similar in both osteogenic media, proliferating cells were detected only in OM (Ki-67 staining). In this medium, also significantly higher levels of calcium on day 28 and a tendency for higher osteogenic marker expression were found. Hence, OM may be superior compared to OKit to support osteogenic induction in AM biomaterial. The media differ in composite concentrations and their FCS source. Additionally, OM is supplemented with vitamin D3. Taken together, these factors may result in the improved performance of OM.

It has previously been described that AM is calcified after implantation in vivo, especially when pretreated with glutaraldehyde. Twelve weeks after s.c. implantation into mice, viable AM stained slightly and glutaraldehyde-fixated, non-viable AM strongly positive by von Kossa [43]. In contrast, we have demonstrated in our in vitro study bone-specific mineralization associated with osteocalcin deposition and upregulation of osteogenic markers under osteogenic conditions for the epithelial as well as mesenchymal layer containing viable cells. Early during culture matrix formation was mostly associated with the epithelial layer. It has also been found that in vitro cultures of isolated hAEC show increased osteogenic differentiation potential compared to hAMSC, however differentiation was less reproducible [44]. Additionally, we have shown previously that in vitro culture of hAEC even for only few passages reduces their osteogenic potential significantly [28]. This loss of function can potentially be circumvented by the strategy of in toto application of viable AM as shown in the present study.

We found a limited increase of osteopontin (SPP1 ) during culture under osteogenic conditions, although dexamethasone and vitamin D3 should stimulate its expression during osteogenesis [45]. However, osteopontin was already expressed at high levels in the native AM material. Osteopontin variants have an impact on a multitude of processes during development, implantation, placentation, as well as biomineralization and exert immunological responses during inflammation and wound healing [46]. Its expression in smooth muscle cells has been reported to inhibit vascular calcification [47]. Due to its versatile function, osteopontin may also play an important regulatory role in AM in term placenta.

In addition to bone matrix formation, high levels of BMP-2 and BMP-4 were induced under osteogenic conditions, although BMP-2 was already expressed in fresh, undifferentiated AM. BMP-2 expression in AM from term placenta in labor, especially in hAEC has been previously reported [48]. Secretion of these growth factors from AM could be relevant in a bone grafting situation, since BMPs are able to induce migration, proliferation of host osteoblasts and endothelial cells [49].

Conclusions

hAEC, major cell population in the AM, show high osteogenic differentiation potential, but are difficult to isolate and expand in vitro [44]. Additionally, they may be phenotypically altered during culture [23, 44] or even loose attributes of sternness during longterm expansion [28]. Furthermore, isolation, long term in vitro expansion and biomaterial seeding is time and cost intensive and increases the risk of contamination with pathogens or of tumorigenic conversion. We have shown in this study that AM per se - with its sessile stem cells - is a suitable biomaterial for osteogenic tissue engineering. It has been demonstrated in this proof of principle in vitro study that the viable biomaterial in toto has the potential to respond to an osteogenic environment. Considering the afore mentioned limitations of in vitro expansion for cell transplants, this novel approach may be a suitable alternative to current bone tissue engineering protocols. Based on preliminary results for chondrogenic differentiation, further investigations into the applicability of this biomaterial for other areas of tissue engineering is suggested.

The present invention is therefore directed to a process for differentiating stem cells within human amniotic membrane by exposing stem cells sessile on the amniotic membrane to osteogenic conditions.

The present invention is also directed to a biomaterial for regenerative medicine obtainable by osteogenic differentiation of stem cells sessile on the human amniotic membrane. A preferred biomaterial accoding to the invention is a grafting material for bone regeneration.

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Figure 1 : Viability of amniotic membranes on day 14 and 28 of culture in control medium (CM), OKit or osteogenic medium (OM): (A) quantified by EZ4U assay and (B) visualized by live/dead staining via CalceinAM/DAPI (green/blue). For EZ4U, means ± SD of % viability relative to fresh AM (dO) from data of 3 individual experiments measured in triplicates are displayed. Significant differences are indicated by asterisks. For live/dead staining one representative AM per condition is displayed. Size bar represents 1000 μηι.

Figure 2: Osteogenic differentiation as evaluated by histological methods. (A) Hematoxylin/eosin staining (HE), von Kossa staining (vK) and Alizarin Red S (AR) staining demonstrating mineralization and immunostaining for osteocalcin (OC) in sections of AM cultured in control medium (CM) or osteogenic media OKit and OM for 14 or 28 days. Pictures of consecutive sections in one representative sector are shown. (B) Detailed view of consecutive sections of AM in CM, OKit and OM (14 and 28 days) stained with vK, and immunostained for activated caspase-3 (Casp3) and proliferation (Ki67).

Figure 5: Osteogenic differentiation as evaluated by histological methods. Hematoxylin/eosin staining (HE), von Kossa staining (vK) and Alizarin Red S (AR) staining demonstrating mineralization and immunostaining for osteocalcin (OC) of fresh AM (dO) and AM cultured in osteogenic media OKit and OM for 14 or 28 days. Overview pictures of consecutive sections in one representative sector are shown. Size bar represents 1000 μηι.

Figure 3: Quantification of (A) calcium and (B) intracellular alkaline phosphatase levels in AM cultured in control medium (CM) or osteogenic media OKit and OM. Mean ± SD of 5 individual experiments measured at least in duplicates are displayed. Significant differences are indicated by asterisks.

Figure 4: Expression levels of the osteogenic markers RUNX2, alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP), BMP-2 and BMP-4 in AM on day 14 and 28 of culture in osteogenic media OKit or OM. qRT-PCR results are normalized to hypoxanthine- guanine phosphoribosyltransferase (HPRT) and presented as fold of induction (FOI) relative to dO expression levels (fresh, uninduced AM). Median and Q1/Q3 of 3 to 6 individual experiments are displayed.

Claims

Claims:

1. Process for differentiating stem cells within human amniotic membrane by exposing stem cells sessile on the amniotic membrane to osteogenic conditions.

2. Biomaterial for regenerative medicine obtainable by osteogenic differentiation of stem cells sessile on the human amniotic membrane.

3. Biomaterial accoding to claim 2, characterized in that it is grafting material for bone regeneration.