Tissue engineering from human mesenchymal amniocytes: a prelude to clinical trials

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Journal of Pediatric Surgery (2007) 42, 974 – 980


Tissue engineering from human mesenchymal amniocytes: a prelude to clinical trials Shaun M. Kunisakia,b, Myriam Armantc, Grace S. Kaoc, Kristen Stevensond, Haesook Kimd, Dario O. Fauzaa,b,* a

Department of Surgery, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA Advanced Fetal Care Center, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA c Center for Human Cell Therapy, CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA 02115, USA d Department of Biostatistics & Computational Biology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA b

Index words: Mesenchymal stem cells; Fetal cells; Amniocytes; Amniotic fluid; Tissue engineering; Cell therapy; Human serum; Fetal bovine serum

Abstract Purpose: The surgical treatment of congenital anomalies using tissues engineered from amniotic fluidderived mesenchymal cells has been validated experimentally. As a prerequisite for testing the clinical feasibility of this therapeutic concept, this study was aimed to expand human mesenchymal amniocytes in the absence of animal products. Methods: Human mesenchymal cells were isolated from amniotic fluid samples (n = 12) obtained at 20 to 37 weeks’ gestation. Their phenotypic profiles and cell proliferation rates were compared during expansion under 2 different media, containing either fetal bovine serum or allogeneic human AB serum. Statistical analyses were by the 2-sided Wilcoxon signed rank test and linear regression ( P b .05). Results: Mesenchymal cells could be isolated and expanded at any gestational age. There was a greater than 9-fold logarithmic expansion of mesenchymal cells, with no significant differences in the overall proliferation rates based on serum type ( P = .94), or gestational age ( P = .14). At any passage, cells expanded for up to 50 days remained positive for markers consistent with a multipotent mesenchymal progenitor lineage, regardless of the medium used. Conclusions: Human mesenchymal amniocytes retain their progenitor phenotype and can be dependably expanded ex vivo in the absence of animal serum. Clinical trials of amniotic fluid-based tissue engineering are feasible within preferred regulatory guidelines. D 2007 Elsevier Inc. All rights reserved.

In addition to their capacity for multilineage differentiation, mesenchymal stem cells (MSCs) have been shown to Papers presented at the 58th Annual Meeting of the Section on Surgery of the American Academy of Pediatrics. * Corresponding author. Children’s Hospital Boston, Boston, MA 02115, USA. Tel.: +1 617 919 2966; fax: +1 617 730 0910. E-mail address: [email protected] (D.O. Fauza). 0022-3468/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2007.01.031

possess remarkable plasticity [1,2]. As a result, over the past decade there has been increasing interest by multiple groups in the use of MSCs in regenerative therapies for various disorders [3-11]. We have recently introduced the notion that the MSCs normally found in the amniotic fluid could be used in tissue engineering strategies for the surgical repair of congenital anomalies in the perinatal period [12,13]. Translated clinically, from a simple amniocentesis, different

Tissue engineering from human mesenchymal amniocytes grafts could be engineered in parallel to the remainder of gestation, so that a child could benefit from having autologous, expanded tissue promptly available for surgical reconstruction, either in the neonatal period or before birth (Fig. 1). Thus far, we have validated this concept in preclinical, large animal models of diaphragmatic and tracheal repairs [14-16]. Before clinical application of mesenchymal amniocytebased fetal tissue engineering, however, we must determine whether these cells can be grown in the absence of animal products in the culture medium. Previous experimental and clinical data have shown that exposure of human cells to fetal bovine serum (FBS), for example, results in fixation of animal proteins on the cell surface, rendering the host more prone to adverse immunemediated and/or inflammatory events, including anaphylactic reactions [17-19]. Moreover, the threat of disease transmission by bacterial, viral, and prion pathogens from the contact with animal-based products remains a valid concern [20]. In fact, the US Food and Drug Administration Agency (FDA) normally defer the approval of novel cell-based therapies that include cell exposure to xenogeneic materials. Until now, the expansion potential and phenotypic characteristics of amniotic MSCs cultured in the absence of FBS remain unknown. This study was aimed at determining the response of human amniotic MSCs when cultured in the absence of animal products and at comparing it with culture under standard FBS-based medium, as a prerequisite for fulfilling the basic regulatory requirements needed for initiation of clinical trials of this therapeutic concept.


1. Materials and methods 1.1. MSC isolation This study was approved by the institutional review board of Children’s Hospital Boston under protocol #S0412-149. Human amniotic fluid specimens (n = 12) were obtained between 20 and 37 weeks’ gestation by amniocentesis or amnioreduction (5.5-500 mL per sample), in a sterile fashion, from fetuses with a normal karyotype. All samples were stored at 48C before further processing at a local, FDA-accredited Good Manufacturing Practice facility. The mesenchymal cell population was then isolated based on methods as previously described by our group [12,21]. Briefly, the amniotic fluid sample was equally divided into 2 tubes and centrifuged at 400 g for 15 minutes. The pellets were then resuspended in growth medium (2 mL medium per 10 mL amniotic fluid) containing either 20% defined FBS (Hyclone, Logan, Utah), or 20% allogeneic pooled human AB serum (ABS) (Cambrex BioScience, Walkersville, Md). The growth medium consisted of high glucose Dulbecco modified Eagle medium with l-glutamine, gentamycin (50 Ag/mL) (all from Cambrex BioScience), and 5 ng/mL basic fibroblast growth factor (Promega, Madison, Wis). Cells were cultured in collagen type I coated 6-well plates (Biocoat, BD Biosciences, San Jose, Calif) and placed in a 5% carbon dioxide incubator at 378C for 7 to 14 days. After 2 days, the nonadherent cells were removed, and 2 mL of fresh media was added to each well.

1.2. MSC proliferation The growth kinetics of the MSCs was studied for up to 50 days in vitro. Briefly, the adherent cells were detached by using 0.05% trypsin/0.53 mmol/L EDTA (Invitrogen Corp, Carlsbad, Calif) when 80% to 90% confluent. Cells were counted and reseeded in growth media containing 20% FBS or 20% ABS (typically 0.5  106 cells in 25 ml in a T162 dish; Corning Inc Life Sciences, Acton, Mass). All subsequent passages were performed similarly. Cell expansion was assessed over time based on the relative days in culture, with day 0 defined as the first passage.

1.3. Flow cytometry

Fig. 1 The clinical concept of amniotic fluid-based fetal tissue engineering for the surgical treatment of congenital anomalies: fetal MSCs isolated from the amniotic fluid are expanded ex vivo and used in an implantable engineered construct either later in gestation, or in postnatal life, for the treatment of a prenatally diagnosed defect.

Fluorescence-activated cell sorting analysis was performed on 7 samples of human mesenchymal amniocytes grown in parallel with either FBS or ABS. Approximately 0.5 to 1  106 cells per staining were incubated with fluorescent-labeled mouse monoclonal antibodies for 30 minutes and then washed twice with PBS. The following antibodies were used: CD9, CD10, CD13, CD29, CD31/PECAM-1, CD44, CD45, CD49a, CD71, CD73 (SH3), CD90/Thy-1, CD106, CD117, HLA-A, B, C (all from BD Biosciences), CD105 (SH2) (eBiosciences, San Diego, Calif), and CD166/ALCAM (Ancell Corp, Bayport, Minn). Nonspecific cell staining was excluded using mouse isotype immunoglobulin controls. The data


S.M. Kunisaki et al.

Fig. 2 Representative morphology of expanded mesenchymal cells from human amniotic fluid under phase-contrast microscopy (original magnification, 40). There were no morphological differences based on the type of serum used in the culture media. HAB indicates human AB serum.

were acquired by using the 6-color BD FACSCanto system (BD Biosciences) and analyzed with FlowJo (Treestar Inc, Ashland, Ore).

1.4. Statistical analysis Statistical analyses were performed by the 2-sided Wilcoxon signed rank test and linear regression, as appropriate ( P b .05).

2. Results Colonies composed of several spindle-shaped, fibroblastoid cells of equivalent size could be successfully isolated and cultured from all the 7 amniotic fluid samples that were processed within 48 hours of procurement. Amniotic fluid specimens processed after 48 hours did not reliably grow mesenchymal cell colonies in either medium. Viable populations of rapidly expanding cells were obtained from samples harvested at any gestational age (mean, 31.6 weeks; range, 20-37 weeks gestation) and from as little as 5.5 mL of amniotic fluid. There were no differences in cell morphology based on the type of culture medium used, at any passage (Fig. 2). Based on a single well containing cells harvested from approximately 10 mL of amniotic fluid, the time required until the first passage was on average 12.1 F 3.1 days. Starting from the first passage, amniotic fluid-derived MSCs were found to proliferate rapidly, regardless of the growth medium used. Overall, there was a greater than 9-fold logarithmic cell expansion in the period studied (mean, 32.9 days), with no significant differences in the overall cell proliferation rates, based on serum type ( P = .94), or gestational age ( P = .14; Fig. 3). Mesenchymal stem cells cultured in FBS appeared to have a smaller variation in growth kinetics compared to those grown in ABS, but this trend was not statistically significant in this initial series. There was no evidence of cell expansion arrest in cultures grown in either FBS or ABS, for up to 38 days after the first passage (a total of up to 50 days in culture).

At any passage, the isolated cells stained consistently positive for several cell surface markers, including CD73 (SH3), CD105 (SH2), CD44, CD29, CD90, CD13, CD10, and CD71, in a profile compatible with a mesenchymal progenitor identity. As expected, these cells also stained positive for HLA-A, B, C, but were negative for CD45, CD34, CD14, CD19, CD8, CD56, and CD31. There were no differences in antigen expression based on gestational age or the type of culture medium used (Fig. 4).

3. Discussion Of all the many sources of MSCs described to date, the amniotic fluid has been increasingly accepted as the ideal one for perinatal regenerative therapies. Our group and others have shown that amniotic fluid-derived MSCs can be

Fig. 3 Graph of the logarithmic cell expansion rates based on the number of days since the first cell passage (relative days). There were no statistical differences in the growth kinetics of amniotic fluid-derived MSCs cultured in FBS when compared to those grown in human ABS ( P N .05).

Tissue engineering from human mesenchymal amniocytes


Fig. 4 Representative ungated flow cytometry analyses of expanded human mesenchymal cells isolated from amniotic fluid. There were no differences in the immunophenotypic profiles of cells grown in FBS (thinner line) compared to those cultured in human ABS (thicker line).

isolated relatively easily, proliferate quickly under standard culture conditions, and have a remarkable multilineage potential [12,13,21-28]. In addition, and perhaps more importantly, a diagnostic amniocentesis is routinely offered to any mother with a fetus in whom a congenital anomaly has been diagnosed by prenatal imaging. A small additional aliquot of amniotic fluid could be effortlessly obtained at that time for the engineering of different tissue grafts, without any further maternal morbidity. Embryonic and fetal cells from the 3 germ layers have long been identified within the amniotic fluid [29-32]. Yet, the fact that progenitor cells can also be found therein was apparently first reported only in 1993, when hematopoietic stem cells were identified there before the 12th week of gestation, possibly coming from the yolk sac [33]. A study from 1996 was the first to suggest the possibility of mutilineage potential of nonhematopoietic cells present in the amniotic fluid, by demonstrating myogenic conversion of select amniocytes [34]. However, that study did not specify the identity of these cells. The presence of mesenchymal cells in the amniotic fluid has been suggested for decades [35,36]. Still, the progenitor identity of mesenchymal amniocytes started to be determined only

very recently [21,37,38]. The amniotic fluid is now known to contain a heterogeneous population of progenitor cells, including, at least, mesenchymal, epithelial, hematopoietic, trophoblastic, and, possibly, even more primitive, embryoniclike stem cells [39]. The cellular profile of the amniotic fluid changes predictably during gestation [33,40]. In addition to its common origin with the mesenchymal portion of the placenta, the amniotic cavity/fluid receives cells shed from the fetus and, quite possibly, from the placenta as well (the latter has yet to be confirmed, though). The mechanisms responsible for the production and turnover of the amniotic fluid are thought to also play a role in the cell types present in the amniotic cavity. Of all the cell types found in the amniotic fluid, the MSCs are the most useful for tissue engineering, in that most of the grafts needed for the surgical repair of congenital anomalies are of a mesenchymal nature. Amniotic MSCs are known to proliferate very quickly in culture, under standard conditions (ie, exposed to FBS). Ovine data have shown that these cells proliferate significantly faster in vitro than immunocytochemically comparable cells derived from fetal or adult subcutaneous connective tissue, neonatal

978 bone marrow, and umbilical cord blood [12,41]. In humans, the expansion potential of mesenchymal amniocytes exceeds that of bone marrow MSCs [13, 38]. The phenotype of human mesenchymal amniocytes expanded in vitro is similar to that reported for MSCs derived from second trimester fetal tissue and adult bone marrow [2,38,42]. In the present study, we have shown that a commercially available serum derived from allogeneic, pooled human donors can be used to reliably isolate and expand amniotic fluid-derived MSCs ex vivo, regardless of gestational age, at rates comparable to that of cells cultured in FBS. Our data also demonstrate that a small sample of amniotic fluid, as little as 5 mL, can easily produce enough cells (ie, more than 100 million) required to engineer a surgically implantable construct in a relatively short period. Our observed increased intersample variability in cell proliferation in the presence of human ABS, however, needs to be further examined, especially in light of the well-known variability among the different lots of this component, as it is commercially offered. Furthermore, whether different/lower concentrations of human serum would produce similar results remains to be demonstrated. Regardless of the serum we used, the mesenchymal amniocytes expressed the exact same markers, which were consistent with a mesenchymal stem/progenitor phenotype, including CD29, CD44, CD90, and CD105. At present, the effect of human serum on these cells’ gene/protein array expression and on their ability to differentiate into the various pathways previously shown under FBS culture remains largely unknown. Early data from 1 study has suggested that the osteogenic differentiation of human bone marrow-derived MSCs is actually enhanced in culture with human ABS, when compared to FBS [43]. Ongoing experiments in our laboratory are currently analyzing the gene expression as well as the multipotent differentiation capacity of amniotic fluid-derived MSCs expanded under human serum. The use of serum derived from an autologous source, or from a parent, represents yet another avenue for further exploration. Although more data should be pursued, the present study shows that human mesenchymal amniocytes retain their progenitor phenotype and can be reliably expanded ex vivo in the absence of animal serum. Combined with our previously reported animal studies, the present data have led to a proposed clinical trial of engineered diaphragmatic repair usingamniotic MSC-based constructs at our institution, currently under review by the FDA. It is to be hoped that other ongoing efforts should lead to similar approaches for the repair of a number of other congenital anomalies in the not too distant future.

Acknowledgment This work was supported by the Center for Human Cell Therapy, as a subaward of the NIH/NHLBI grant no. U24 HL074355-01A1. SMK was supported by grants from the

S.M. Kunisaki et al. NIH National Research Service Award DK065406-02 and the VH Kazanjian Surgical Research Fellowship of the Massachusetts General Hospital.

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Discussion Peter Kim, MD (Toronto, Canada): What’s the origin of these stem cells and how do you propose to regulate the characteristics including the way in which they proliferate? Kunisaki, MD (response): With regard to the origin of these, the short answer is it’s entirely unclear. We speculate, however, that these are again of mesenchymal cells that are likely from the fetal portion of the placenta. And we think they may be involved in either the healing process along the maternal fetal interface with the placenta. But that’s really sort of speculation. The amniotic fluid contains a myriad of different types of cells. And the cells that we specifically were looking at were the mesenchymal population. With regard to the actual growth of these cells, the only data we have thus far are from our large animal in vivo models, which show that these cells do have a growth potential eventually when implanted in the proper conditions. But certainly, in vitro they have a capacity to grow to actually greater than 40 passages under the proper conditions of which fetal bovine serum has been included. Darrel Cass, MD (Houston, Tex): In a practical sense, what are you proposing that we’re going to do with these cells? In the past studies that I’ve seen from your group, we’ve seen a high failure rate in using AlloDerm, for example, alone in trying to reconstruct the diaphragm. But I think that was done in the past tome when there was just a thin AlloDerm available. And now that there’s thicker and different products, I’m not so sure that the failure rate is going to be as high with just that tissue alone, with the AlloDerm alone and the scaffold alone. So what are you going to do with these cells now that you’ve been able to isolate them? Kunisaki, MD (response): In addition to congenital diaphragmatic hernia, there are a myriad of other potential applications as this forum in general has suggested. I think the potential therapies for mesenchymal stem cells, whether they be derived from the amniotic fluid, the bone marrow, umbilical cord blood, etc, really might be used to do many things, either to facilitate better wound healing and in terms of actually trying to create a cell source for an engineered graft. And with regard to your question or speculation about the failure of AlloDerm, our study primarily addressed that we do think that cell seeded AlloDerm is much better than AlloDerm in general. And this was tested not obviously in a human model, but in a very rigorous ovine model in which a neonatal lamb becomes a fully grown sheep in the span of a year. And so you get essentially 100% failure rate with AlloDerm alone and on the order of only 25% in this very rigorous model where the cells have to adapt and


S.M. Kunisaki et al. the extracellular matrix has to adapt in a very short time period. So I’m still very optimistic given the fact that the current at least quoted recurrence rate for a diaphragmatic hernia is anywhere from 25% to 50%. And I am optimistic that these cells may have a role in terms of lowering that complication rate.

Karl Sylvester, MD (Palo Alto, Calif): Can you elaborate a little about what the clinical strategy is? Meaning since these cells all have HLA antigen expression, is it going to be to transplant them to patients in an autologous fashion or allogeneically? And if it’s autologous, obviously you would have to have that information prenatally, which presumptively ultrasound would supply. And along those same lines, since the cells are so highly proliferative in vitro, have you analyzed the karyotype of these after expansion, since there’s probably some instability to that through serial passage? Kunisaki, MD (response): I think our strategy initially will be to do this in an autologous fashion as you suggested,

although we have not completely discounted the potential of an allogeneic graft using these cells. There is at least increasing data in the literature, predominantly again from bone marrow derived mesenchymal stem cells, that they may have immunosuppressive or immunoregulatory properties. And these are in studies that are primarily interested in using them to facilitate hematopoietic engraftment across allogeneic barriers. Certainly, there is increasing evidence, predominantly from Europe, that shows that amniotic fluid based mesenchymal stem cells may have similar immunoregulatory properties. But I think really the verdict is still out on that and certainly further work needs to be done. But I do think that we’re much closer. And you know this also, when you think about it, could also apply to problems later in life. For example, we have shown from this study that only about 5 mL of amniotic fluid is required to isolate these cells. And you can envision for example that these cells could be banked for future use. And that would be a very easily attainable way.

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