In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage.

Gekas J, Walther G, Skuk D, Bujold E, Harvey I, Bertrand OF

Clin Exp Med. 2009 Sep 3. [Epub ahead of print]
In vitro and in vivo study of human amniotic fluid-derived stem cell differentiation into myogenic lineage.

Gekas J, Walther G, Skuk D, Bujold E, Harvey I, Bertrand OF.

Laboratoire de Génétique Humaine, Centre Hospitalier de l'Université Laval, Quebec City, QC, Canada.

Recent findings have shown that amniotic fluid (AF) could be a putative new source of multipotent stem cells (SC). We investigated whether these human SC could efficiently differentiate into myogenic lineage in vitro and integrate in vivo skeletal muscle in severe combined immunodeficiency (SCID) mice. C/kit immunomagnetic-sorted AF (AF c/kit+) SC were characterized by immunocytochemistry and Southern blotting for myogenic markers (desmin, MyoD). In vitro, AF c/kit+ SC phenotypic conversion into myogenic cells was assayed by myogenic-specific induction media. AF c/kit+ SC without ex vivo manipulation were transplanted into the tibialis anterior (TA) of (SCID) mice. Acquisition of a myogenic-like phenotype (desmin, MyoD) in AF c/kit+ SC was observed after culture in myogenic-specific induction media. In vivo, transplanted AF c/kit+ SC showed an engraftment in the skeletal muscle of SCID mice, but with unexpected tubular glandular tissue-like differentiation. Importantly, no immuno-rejection, inflammatory response or tumorigenicity of these cells was found. Within these experimental conditions, AF c/kit+ SC were able to differentiate into myogenic cells in vitro, but not in vivo after their transplantation into the skeletal muscle of SCID mice. Because AF c/kit+ SC survived and differentiated into tubular gland-like cells after their transplantation in the TA, an ex vivo engagement in myogenic pathway prior their transplantation could favor their differentiation into myogenic cells in vivo.


J Biomed Sci. 2009 Aug 23;16:75.
Escalated regeneration in sciatic nerve crush injury by the combined therapy of human amniotic fluid mesenchymal stem cells and fermented soybean extracts, Natto.

Pan HC, Yang DY, Ho SP, Sheu ML, Chen CJ, Hwang SM, Chang MH, Cheng FC.

Department of Neurosurgery, Taichung Veterans General Hospital, Taiwan, Republic of China. Hcpan2003@yahoo.com.tw

Attenuation of inflammatory cell deposits and associated cytokines prevented the apoptosis of transplanted stem cells in a sciatic nerve crush injury model. Suppression of inflammatory cytokines by fermented soybean extracts (Natto) was also beneficial to nerve regeneration. In this study, the effect of Natto on transplanted human amniotic fluid mesenchymal stem cells (AFS) was evaluated. Peripheral nerve injury was induced in SD rats by crushing a sciatic nerve using a vessel clamp. Animals were categorized into four groups: Group I: no treatment; Group II: fed with Natto (16 mg/day for 7 consecutive days); Group III: AFS embedded in fibrin glue; Group IV: Combination of group II and III therapy. Transplanted AFS and Schwann cell apoptosis, inflammatory cell deposits and associated cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. The deterioration of neurological function was attenuated by AFS, Natto, or the combined therapy. The combined therapy caused the most significantly beneficial effects. Administration of Natto suppressed the inflammatory responses and correlated with decreased AFS and Schwann cell apoptosis. The decreased AFS apoptosis was in line with neurological improvement such as expression of early regeneration marker of neurofilament and late markers of S-100 and decreased vacuole formation. Administration of either AFS, or Natto, or combined therapy augmented the nerve regeneration. In conclusion, administration of Natto may rescue the AFS and Schwann cells from apoptosis by suppressing the macrophage deposits, associated inflammatory cytokines, and fibrin deposits.


Stem Cells Dev. 2009 Aug 17. [Epub ahead of print]
Secretory Profiles and Wound Healing Effects of Human Amniotic Fluid-derived Mesenchymal Stem Cells.

Yoon BS, Moon JH, Jun EK, Kim J, Maeng I, Kim JS, Lee JH, Baik CS, Kim A, Cho KS, Lee JH, Lee HH, Whang KY, You S.

Korea University, Seoul, Korea, Republic of; biosun302@hanmail.net.

Recent evidence shows that amniotic fluid contains multiple cell types derived from the developing fetus, and may represent a novel source of stem cells for cell therapy. In this study, we examined the paracrine factors released by human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) and their ability to accelerate the wound healing process by stimulating proliferation and migration of dermal fibroblasts. AF-MSCs expressed the typical mesenchymal stem cell marker proteins CD13, CD29, and CD44 and differentiated into adipocytes, osteoblasts and chondrocytes when exposed to the appropriate differentiation media. In addition, AF-MSC-conditioned media (AF-MSC-CM) significantly enhanced proliferation of dermal fibroblasts. Antibody-based protein array and ELISA indicated that AF-MSC-CM contains various cytokines and chemokines that are known to be important in normal wound healing, including IL-8, IL-6, TGF-beta, TNFRI, VEGF, and EGF. Application of AF-MSC-CM significantly enhanced wound healing by dermal fibroblasts via the TGF-beta/SMAD2 pathway. Levels of p-SMAD2 were increased by AF-MSC-CM, and both the increase in p-SMAD2 and migration of dermal fibroblasts were blocked by inhibiting the TGF-beta/SMAD2 pathway. Moreover, in a mouse excisional wound model, AF-MSC-CM accelerated wound healing. These data provide the first evidence of the potential for AF-MSC-CM in the treatment of skin wounds.

PMID: 19686050 [PubMed - as supplied by publisher]


5: Hum Mol Genet. 2009 Aug 13. [Epub ahead of print+
Pluripotency can be rapidly and efficiently induced in human amniotic fluid-derived cells.

Li C, Zhou J, Shi G, Ma Y, Yang Y, Gu J, Yu H, Jin S, Wei Z, Chen F, Jin Y.

Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine; 225 South Chongqing Road, Shanghai, 200025, China.

Direct reprogramming of human somatic cells into pluripotency has broad implications in generating patient-specific induced pluripotent stem (iPS) cells for disease modeling and cellular replacement therapies. However, the low efficiency and safety issues associated with generation of human iPS cells have limited their usage in clinical settings. Cell types can significantly influence reprogramming efficiency and kinetics. To date, human iPS cells have been obtained only from a few cell types. Here, we report for the first time rapid and efficient generation of iPS cells from human amniotic fluid-derived cells (hAFDCs) via ectopic expression of four human factors: OCT4/SOX2/KLF4/C-MYC. Significantly, typical single iPS cell colonies can be picked up six days after viral infection with high efificiency. Eight iPS cell lines have been derived. They can be continuously propagated in vitro and express pluripotency markers such as AKP, OCT4, SOX2, SSEA4, TRA-1-60 and TRA-1-81, maintaining the normal karyotype. Transgenes are completely inactivated and the endogenous OCT4 promoter is adequately demethylated in the established iPS cell lines. Moreover, various cells and tissues from all three germ layers are found in embryoid bodies and teratomas respectively. In addition, microarray analysis demonstrates a high correlation coefficient between hAFDC-iPS cells and human embryonic stem cells, but a low correlation coefficient between hAFDCs and hAFDC-iPS cells. Taken together, these data identify an ideal human somatic cell resource for rapid and efficient generation of iPS cells, allowing us to establish human iPS cells using more advanced approaches and possibly to establish disease- or patient-specific iPS cells.

PMID: 19679563 [PubMed - as supplied by publisher]
________________________________________

8: Cytotherapy. 2009;11(5):534-47.
Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels.

Mareschi K, Rustichelli D, Comunanza V, De Fazio R, Cravero C, Morterra G, Martinoglio B, Medico E, Carbone E, Benedetto C, Fagioli F.

Stem Cell Transplantation and Cellular Therapy Unit, Pediatric Onco-Hematology Department, Regina Margherita Children's Hospital, Turin, Italy. Katia.mareschi@unito.it

BACKGROUND AIMS: Amniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro. METHODS: AF was harvested during routine pre-natal amniocentesis at 14-16 weeks of pregnancy. AF sample pellets were plated in alpha-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction. RESULTS: Twenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na(+) channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na(+) channels. CONCLUSIONS: These data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.


________________________________________
Eur Rev Med Pharmacol Sci. 2009 Mar;13 Suppl 1:71-8.
Mesenchymal stromal cells multipotency and plasticity: induction toward the hepatic lineage.

Saulnier N, Lattanzi W, Puglisi MA, Pani G, Barba M, Piscaglia AC, Giachelia M, Alfieri S, Neri G, Gasbarrini G, Gasbarrini A.

Department of Internal Medicine and Gastroenterology, Catholic University of the Sacred Heart, Rome, Italy. nathaliesaulnier@yahoo.fr

BACKGROUND: Human mesenchymal stromal cells (MSCs) can be isolated from a variety of adult and perinatal tissues and exert multipotency and self renewal properties which make them suitable for cell-based therapy. Their potential plasticity extended to non-mesodermal-derived tissues has been indicated, although it is still a debated issue. In this study we have isolated MSCs from both adult and fetal tissues. Their growth, immunophenotype and multi-lineage differentiation potentials have been analyzed, focusing, in particular, on the hepatic differentiation. METHODS: Cells were isolated from bone marrow (BMSC), adipose tissue (ATSC) and second trimester amniotic fluid (AFSC), upon a written informed consent obtained from donor patients. Cells were expanded and growth kinetics was assessed by means of proliferation assay. Their immunophenotype was analyzed using cytometry and multi-lineage differentiation potential was evaluated by means of in vitro differentiation assays. Finally, the expression of tissue-specific markers was also assessed by mean of semi-quantitative PCR. RESULTS: Bipolar spindle-shaped cells were successfully isolated from all these tissues. Interestingly, ATSCs and AFSCs showed a higher proliferation potential than BMSCs. Mesodermal differentiation capacity was verified in all MSC populations, even if AFSCs were not able to undergo adipogenesis in our culture conditions. Furthermore, we showed that MSC cultured in appropriate conditions were able to induce hepatic-associated genes, such as ALB and TDO2. CONCLUSION: Taken together the data here reported suggest that MSCs from both adult and fetal tissues are capable of tissue-specific commitment along mesodermal and non-mesodermal lineages. In particular we have demonstrated that a specific hepatogenic commitment can be efficiently induced, proposing these cells as suitable tool for cell-based applications aimed at liver regeneration.

________________________________________
10: J Pediatr Surg. 2009 Jun;44(6):1120-6; discussion 1126.
Sternal repair with bone grafts engineered from amniotic mesenchymal stem cells.

Steigman SA, Ahmed A, Shanti RM, Tuan RS, Valim C, Fauza DO.

Department of Surgery, Children's Hospital Boston and Harvard Medical School, Boston, MA 02115, USA.

PURPOSE: We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be used in postnatal sternal repair. METHODS: Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n = 6). Constructs were dynamically maintained in an osteogenic medium and equally divided into 2 groups with respect to time in vitro as follows: 14.6 or 33.9 weeks. They were then used to repair full-thickness sternal defects spanning 2 to 3 intercostal spaces in allogeneic kits (n = 6). Grafts were submitted to multiple analyses 2 months thereafter. RESULTS: Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by microcomputed tomographic scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements preimplantation and postimplantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the 2 construct groups in any comparison. CONCLUSIONS: Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction.

________________________________________
11: Stem Cells Dev. 2009 Jun 9. [Epub ahead of print]

Endothelial Differentiation of Amniotic Fluid-derived Stem Cells: Synergism of biochemical and shear force stimuli.

Zhang P, Baxter J, Vinod K, Tulenko TN, Dimuzio P.

Thomas Jefferson University, Surgery, Philadelphia, Pennsylvania, United States; ping.zhang@jefferson.edu.

Human amniotic fluid-derived stem (AFS) cells possess several advantages over embryonic and adult stem cells, as evidenced by expression of both types of stem cell markers and ability to differentiate into cells of all three germ layers. Herein, we examine endothelial differentiation of AFS in response to growth factors, shear force and hypoxia. We isolated human AFS from amniotic fluid samples (1-4cc/specimen) obtained from patients undergoing amniocentesis at 15-18 weeks of gestation (n=10). Isolates maintained in non-differentiating medium expressed the stem cell markers CD13, CD29, CD44, CD90, CD105, OCT4 and SSEA-4 through passage eight. After three weeks of culture in Endothelial Growth Media (EGM2), the stem cells exhibited an endothelial-like morphology, formed cord-like structures when plated on Matrigel, and up took acetylated-LDL/lectin. Additionally, mRNA and protein levels of CD31 and von Willebrand factor (vWF) significantly increased in response to culture in EGM2, with further up-regulation when stimulated by physiological levels (12 dyne/cm2) of shear force. Culture in hypoxic conditions (5% O2) resulted in significant expression of Vascular Endothelial Growth Factor (VEGF) and Placental Growth Factor (PGF) mRNA. This study suggests that AFS, isolated from minute amounts of amniotic fluid, acquire endothelial cell characteristics when stimulated by growth factors and shear force, and produce angiogenic factors (VEGF, PGF and HGF) in response to hypoxia. Thus, amniotic fluid represents a rich source of mesenchymal stem cells potentially suitable for use in cardiovascular regenerative medicine.

________________________________________
12: Curr Stem Cell Res Ther. 2009 Sep 1. [Epub ahead of print]
Related Articles, Links

Lost in Translation: What is Limiting Cardiomyoplasty and Can Tissue Engineering Help?

Simpson D, Dudley SC Jr.

Section of Cardiology, University of Illinois at Chicago, 840 S. Wood Street, MC 715, Chicago, IL 60612, USA. scdudley@uic.edu.

Heart failure accounts for more deaths in the United States than any other detrimental human pathology. Recently, repairing the heart after seemingly irreversible injury leading to heart failure appears to have come within reach. Cellular cardiomyoplasty, transplanting viable cell alternatives into the diseased myocardium, has emerged as a promising possible solution. Translating this approach from the laboratory to the clinic, however, has been met with several challenges, leaving many questions unanswered. This review assesses the state of investigation of several progenitor cell sources, including induced pluripotent stem cells, embryonic stem cells, bone marrow stem cells, adipose-derived adult stem cells, amniotic fluid stem cells, skeletal muscle progenitors, induced pluripotent stem cells and cardiac progenitors. Several current roadblocks to maximum success are discussed. These include understanding the need for cardiomyocyte differentiation, appreciating the role of paracrine factors, and addressing the low engraftment rates using current techniques. Tissue engineering strategies to address these obstacles and to help maximize cellular cardiomyoplasty success are reviewed.

PMID: 19492979 [PubMed - as supplied by publisher]
________________________________________
13: Proc Natl Acad Sci U S A. 2009 Jun 16;106(24):9826-30. Epub 2009 May 29.
Induced pluripotent stem cells offer new approach to therapy in thalassemia and sickle cell anemia and option in prenatal diagnosis in genetic diseases.

Ye L, Chang JC, Lin C, Sun X, Yu J, Kan YW.

Department of Medicine, University of California, San Francisco, CA 94143-0793, USA.

The innovation of reprogramming somatic cells to induced pluripotent stem cells provides a possible new approach to treat beta-thalassemia and other genetic diseases such as sickle cell anemia. Induced pluripotent stem (iPS) cells can be made from these patients' somatic cells and the mutation in the beta-globin gene corrected by gene targeting, and the cells differentiated into hematopoietic cells to be returned to the patient. In this study, we reprogrammed the skin fibroblasts of a patient with homozygous beta(0) thalassemia into iPS cells, and showed that the iPS cells could be differentiated into hematopoietic cells that synthesized hemoglobin. Prenatal diagnosis and selective abortion have been effective in decreasing the number of beta-thalassemia births in some countries that have instituted carrier screening and genetic counseling. To make use of the cells from the amniotic fluid or chorionic villus sampling that are used for prenatal diagnosis, we also showed that these cells could be reprogrammed into iPS cells. This raises the possibility of providing a new option following prenatal diagnosis of a fetus affected by a severe illness. Currently, the parents would choose either to terminate the pregnancy or continue it and take care of the sick child after birth. The cells for prenatal diagnosis can be converted into iPS cells for treatment in the perinatal periods. Early treatment has the advantage of requiring much fewer cells than adult treatment, and can also prevent organ damage in those diseases in which damage can begin in utero or at an early age.

________________________________________
14: Catheter Cardiovasc Interv. 2009 Jun 1;73(7):917-24.
Amniotic stem cells for cellular cardiomyoplasty: promises and premises.

Walther G, Gekas J, Bertrand OF.

Research Center, Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie, Québec, Canada.

Cellular cardiomyoplasty is undergoing intensive investigation as a new form of therapy for severely damaged hearts. Among several cell types, mesenchymal stem cells (MSCs) have been proposed as a potential cell source. MSC can be found in adult tissues or in fetal tissues like the umbilical chord blood, amniotic membrane, or amniotic fluid (AF). AF-MSCs have properties intermediate between embryonic and adult MSC, which make them particularly attractive for cellular regeneration. It has been shown that MSC could differentiate in cardiomyocytes-like cells in vitro. In some animal models, it has also been shown that transplanted MSC could engraft and show some cardiomyocytes-like characteristics. Since MSC do not express HLA-DR and present in vitro and in vivo immunosuppressive properties, they can be envisioned to be used in allogenic cellular cardiomyoplasty. Based on these promises, MSC from adult donors are currently used in small safety and feasibility trials. No clinical trial using AF-MSC has been performed yet. Still, the exact role of true cell repopulation and in situ cardiomyocytes differentiation versus pure paracrine effect after cell transplantation is currently much debated. Cellular cardiomyoplasty is a fascinating new area of investigation in regenerative medicine. Although considerable knowledge has been gained over the last decade on the use of MSC as a potential stem cell (SC) source, many issues remain unsolved. Because of several limitations in animal models, clinical studies in highly selected patients balancing the risks and benefits are required. In that regard, MSCs obtained from the fetal AF are a potential new source of SCs that need to be further investigated for cellular cardiomyoplasty. (c) 2009 Wiley-Liss, Inc.


________________________________________
15: Regen Med. 2009 May;4(3):423-33.
Related Articles, Links

Novel sources of fetal stem cells: where do they fit on the developmental continuum?

Pappa KI, Anagnou NP.

First Department of Obstetrics & Gynecology, University of Athens School of Medicine, Greece. kpappa@med.uoa.gr

The recent isolation of fetal stem cells from several sources either at the early stages of development or during the later trimesters of gestation, sharing similar growth kinetics and expressing pluripotency markers, provides strong support to the notion that these cells may be biologically closer to embryonic stem cells, actually representing intermediates between embryonic stem cells and adult mesenchymal stem cells, regarding proliferation rates and plasticity features, and thus able to confer an advantage over postnatal mesenchymal stem cells derived from conventional adult sources such as bone marrow. This conclusion has been strengthened by the different pattern of growth potential between the two stage-specific types of sources, as assessed by transcriptomic and proteomic analysis. A series of recent studies regarding the numerous novel features of fetal stem cells has reignited our interest in the field of stem-cell biology and in the possibilities for the eventual repair of damaged organs and the generation of in vitro tissues on biomimetic scaffolds for transplantation. These studies, employing elegant approaches and novel technologies, have provided new insights regarding the nature and the potential of fetal stem cells derived from placenta, amniotic fluid, amnion or umbilical cord. In this update, we highlight the major progression that has occurred in fetal stem-cell biology and discuss the most important areas for future investigation in the field of regenerative medicine.

Publication Types:
• Research Support, Non-U.S. Gov't
• Review

PMID: 19438317 [PubMed - indexed for MEDLINE]
________________________________________
19: Tissue Eng Part A. 2009 Apr 5. [Epub ahead of print]
Amniotic Fluid Stem Cells Produce Robust Mineral Deposits on Biodegradable Scaffolds.

Peister A, Deutsch ER, Kolambkar Y, Hutmacher DW, Guldberg R.

Morehouse College, Biology, 830 Westview Drive SW, Altanta, Georgia, United States, 30314, 404-653-7879, 404-507-8627; apeister@morehouse.edu.

Bone regeneration through cell-based therapies is limited by insufficient availability of osteogenic cells. This study investigated the potential of amniotic fluid-derived stem cells (AFS cells) to synthesize mineralized extracellular matrix within porous medical-grade poly-epsilon-caprolactone (mPCL) scaffolds. The AFS cells were initially differentiated in 2D culture to determine appropriate osteogenic culture conditions and verify physiologic mineral production by the AFS cells. The AFS cells were then cultured on 3D mPCL scaffolds (6 mm diameter x 9 mm height) and analyzed for their ability to differentiate to osteoblastic cells in this environment. The amount and distribution of mineralized matrix production was quantified throughout the mPCL scaffold using nondestructive microCT analysis and confirmed through biochemical assays. Sterile microCT scanning provided longitudinal analysis of long-term cultured mPCL constructs to determine the rate and distribution of mineral matrix within the scaffolds. The AFS cells deposited mineralized matrix throughout the mPCL scaffolds and remained viable after 15 weeks of 3D culture. The effect of pre-differentiation of the AFS cells on the subsequent bone formation in vivo was determined in a rat subcutaneous model. Cells that were pre-differentiated for 28 days in vitro produced a 7-fold increase in mineralized matrix when implanted subcutaneously in vivo. This study demonstrated the potential of AFS cells to produce 3D mineralized bioengineered constructs in vitro and in vivo and suggests that AFS cells may be an effective cell source for functional repair of large bone defects.

PMID: 19344289 [PubMed - as supplied by publisher]
________________________________________
20: Regen Med. 2009 Mar;4(2):275-91.
Amniotic membrane and amniotic fluid-derived cells: potential tools for regenerative medicine?

Parolini O, Soncini M, Evangelista M, Schmidt D.

Centro di Ricerca E Menni, Fondazione Poliambulanza - Istituto Ospedaliero, Via Bissolati, 57, 25124 Brescia, Italy. ornella.parolini@tin.it

Human amniotic membranes and amniotic fluid have attracted increasing attention in recent years as a possible reserve of stem cells that may be useful for clinical application in regenerative medicine. Many studies have been conducted to date in terms of the differentiation potential of these cells, with several reports demonstrating that cells from both the amniotic fluid and membrane display high plasticity. In addition, cells from the amniotic membrane have also been shown to display immunomodulatory characteristics both in vivo and in vitro, which could make them useful in an allotransplantation setting. Here, we provide an overview comparing the latest findings regarding the stem characteristics of cells from both the amniotic membrane and amniotic fluid, as well as on the potential utility of these cells for future clinical application in regenerative medicine.



Hum Immunol. 2009 Jan;70(1):16-23. Epub 2008 Nov 14.
Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells.

Roelen DL, van der Mast BJ, in't Anker PS, Kleijburg C, Eikmans M, van Beelen E, de Groot-Swings GM, Fibbe WE, Kanhai HH, Scherjon SA, Claas FH.

Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, The Netherlands. D.L.Roelen@LUMC.nl

Protective mechanisms are likely to be present at the fetomaternal interface because fetus-specific alloreactive T cells present in the decidua do not harm the fetus. We tested the immunosuppressive capacity of maternal and fetal multipotent stromal cells (MSC). Single cell suspensions were made from second-trimester amnion, amniotic fluid, and decidua. Culture-expanded cells were identified as MSC based on phenotype and multilineage potential. Coculture of MSC in a primary mixed lymphocyte culture of unrelated responder-stimulator combinations resulted in a dose-dependent inhibition of proliferation. Fetal MSC demonstrated a significantly higher inhibition compared with maternal MSC. This stronger inhibition by fetal MSC was even more prominent in a secondary mixed lymphocyte reaction (MLR) with primed alloreactive T cells. Analysis of cytokine production revealed that fetal MSC produced significantly more interleukin (IL)-10 and vascular endothelial growth factor than maternal MSC. Cell-cell contact is needed for part of the inhibitory effects of MSC. In addition, soluble factors play a role because blocking experiments with anti-IL-10 revealed that the inhibition of the MLR response by fetal MSC is mainly mediated by IL-10. For maternal MSC, other soluble factors seem to be involved. Fetal MSC derived from the fetomaternal interface have a stronger inhibitory effect on naive and antigen-experienced T cells compared with maternal MSC, which is probably related to their higher IL-10 production.




Blood. 2009 Apr 23;113(17):3953-60. Epub 2009 Feb 12.
Human and murine amniotic fluid c-Kit+Lin- cells display hematopoietic activity.
Ditadi A, de Coppi P, Picone O, Gautreau L, Smati R, Six E, Bonhomme D, Ezine S, Frydman R, Cavazzana-Calvo M, André-Schmutz I.

Inserm U768, Paris, France.

We have isolated c-Kit(+)Lin(-) cells from both human and murine amniotic fluid (AF) and investigated their hematopoietic potential. In vitro, the c-Kit(+)Lin(-) population in both species displayed a multilineage hematopoietic potential, as demonstrated by the generation of erythroid, myeloid, and lymphoid cells. In vivo, cells belonging to all 3 hematopoietic lineages were found after primary and secondary transplantation of murine c-Kit(+)Lin(-) cells into immunocompromised hosts, thus demonstrating the ability of these cells to self-renew. Gene expression analysis of c-Kit(+) cells isolated from murine AF confirmed these results. The presence of cells with similar characteristics in the surrounding amnion indicates the possible origin of AF c-Kit(+)Lin(-) cells. This is the first report showing that cells isolated from the AF do have hematopoietic potential; our results support the idea that AF may be a new source of stem cells for therapeutic applications.


BMC Biotechnol. 2009 Feb 16;9:9.
Isolation of osteogenic progenitors from human amniotic fluid using a single step culture protocol.

Antonucci I, Iezzi I, Morizio E, Mastrangelo F, Pantalone A, Mattioli-Belmonte M, Gigante A, Salini V, Calabrese G, Tetè S, Palka G, Stuppia L.

Department of Biomedical Sciences, G, d'Annunzio University, Chieti-Pescara, Italy. antonucciivana@libero.it

BACKGROUND: Stem cells isolated from amniotic fluid are known to be able to differentiate into different cells types, being thus considered as a potential tool for cellular therapy of different human diseases. In the present study, we report a novel single step protocol for the osteoblastic differentiation of human amniotic fluid cells. RESULTS: The described protocol is able to provide osteoblastic cells producing nodules of calcium mineralization within 18 days from withdrawal of amniotic fluid samples. These cells display a complete expression of osteogenic markers (COL1, ONC, OPN, OCN, OPG, BSP, Runx2) within 30 days from withdrawal. In order to test the ability of these cells to proliferate on surfaces commonly used in oral osteointegrated implantology, we carried out cultures onto different test disks, namely smooth copper, machined titanium and Sandblasted and Acid Etching titanium (SLA titanium). Electron microscopy analysis evidenced the best cell growth on this latter surface. CONCLUSION: The described protocol provides an efficient and time-saving tool for the production of osteogenic cells from amniotic fluid that in the future could be used in oral osteointegrated implantology.



Rev Assoc Med Bras. 2008 Nov-Dec;54(6):489-93.
[Isolation, differentiation and biochemical aspects of amniotic fluid stem cell]

[Article in Portuguese]

Cabral AC, Angelo PC, Leite HV, Pereira AK, Lopes AP, de Oliveira MB, Borges KB, Pardini VC, de Sousa Ferreira AC.

Departamento de Genética Humana e pela Criovida do Instituto H. Pardini, Hospital das Clínicas, Faculdade de Farmácia da Universidade Federal de Minais Gerais, Belo Horizonte, MG.

The mesenchymals stem cells (MSCs) are cells with the great potential of differentiation are being introduced in the clinic for treatment of several diseases. Mesenchymal stem cells have several advantages including the stability of their phenotype in vitro. BACKGROUND: isolation of MSCs in amniotic fluid, its expansion and the demonstration of the capacity of these cells to differentiate in adipogenic and myogenic cells, without to change the chromosomal stability of the MSCs in culture. METHODS: in order to evaluate the functional change of these cells, were gotten values of the differentiated adipogenic cells and not differentiated through the dosage of triglycerides. The myogenic nature of the differentiated cells was analyzed comparing the creatine kinase--CK, lactic dehydrogenase--LDH and aldolase produced by the cells. RESULTS: the values of triglycerides were significantly higher in differentiated cells, showing intracytoplasmatic granule form after differentiation. All the biochemical characters were significantly higher in differentiated myogenic cells. CONCLUSIONS: this study suggests that the standardized protocol of differentiation can be used in the attainment of cells with characteristics of adipogenic and muscular cells, from amniotic fluid.



J Cell Biochem. 2009 Mar 1;106(4):507-11.
Stem cell sources to treat diabetes.

Furth ME, Atala A.

Department of Urology and Wake Forest, Institute for Regenerative Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA.

We review progress towards the goal of utilizing stem cells as a source of engineered pancreatic beta-cells for therapy of diabetes. Protocols for the in vitro differentiation of embryonic stem (ES) cells based on normal developmental cues have generated beta-like cells that produce high levels of insulin, albeit at low efficiency and without full responsiveness to extracellular levels of glucose. Induced pluripotent stem (iPS) cells also can yield insulin-producing cells following similar approaches. An important recent report shows that when transplanted into mice, human ES-derived cells with a phenotype corresponding to pancreatic endoderm matured to yield cells capable of maintaining near-normal regulation of blood sugar [Kroon et al., 2008]. Major hurdles that must be overcome to enable the broad clinical translation of these advances include teratoma formation by ES and iPS cells, and the need for immunosuppressive drugs. Classes of stem cells that can be expanded extensively in culture but do not form teratomas, such as amniotic fluid-derived stem cells and hepatic stem cells, offer possible alternatives for the production of beta-like cells, but further evidence is required to document this potential. Generation of autologous iPS cells should prevent transplant rejection, but may prove prohibitively expensive. Banking strategies to identify small numbers of stem cell lines homozygous for major histocompatibility loci have been proposed to enable beneficial genetic matching that would decrease the need for immunosuppression.


43: Int J Immunopathol Pharmacol. 2008 Jul-Sep;21(3):595-602.
Potential role of culture mediums for successful isolation and neuronal differentiation of amniotic fluid stem cells.

Orciani M, Emanuelli M, Martino C, Pugnaloni A, Tranquilli AL, Di Primio R.

Department of Molecular Pathology and Innovative Therapies, Histology Section, Marche Polytechnic University, Ancona, Italy.

In recent years, the use of stem cells has generated increasing interest in regenerative medicine and cancer therapies. The most potent stem cells derive from the inner cell mass during embryonic development and their use yields serious ethical and methodological problems. Recently, a number of reports suggests that another suitable source of multipotent stem cells may be the amniotic fluid. Amniotic fluid mesenchymal stem cells (AFMSCs) are capable of extensive self-renewal, able to differentiate in specialized cells representative of all three germ layers, do not show ethical restriction, and display minimal risks of teratomas and a very low immunogenity. For all these reasons, amniotic fluid appears as a promising alternative source for stem cell therapy. Their recent discovery implies a lack of knowledge of their specific features as well as the existence of a protocol universally recognized as the most suitable for their isolation, growth and long-term conservation. In this study, we isolated stem cells from six amniotic fluids; these cells were cultured with three different culture mediums (Mesenchymal Stem Cell Medium (MSCGM), PC-1 and RPMI-1640), characterized by cytofluorimetric analysis, and then either frozen or induced to neuronal differentiation. Even if the immunophenotype seemed not to be influenced by culture medium (all six samples cultured in the above-mentioned mediums expressed surface antigens commonly found on stem cells), cells showed different abilities to differentiate into neuron-like cells and to re-start the culture after short/long-term storage. Cells isolated and cultured in MSCGM showed the highest proliferation rate, and formed neuron-like cells when sub-plated with neuronal differentiation medium. Cells from PC-1, on the contrary, displayed an increased ability to re-start culture after short/long term storage. Finally, cells from RPMI-1640, even if expressing stem cells markers, were not able to differentiate in neuron-like cells. Further studies are still needed in order to assess the effective role of culture medium for a successful isolation, growth, differentiation and storage of AFMSCs, but our data underline the importance of finding a universally accepted protocol for the use of these cells.