In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment.

In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)-derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM-HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene-engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. Stem Cells 2019;37:1176-1188.

Decellularized Diaphragmatic Muscle Drives a Constructive Angiogenic Response In Vivo.

Skeletal muscle tissue engineering (TE) aims to efficiently repair large congenital and acquired defects. Biological acellular scaffolds are considered a good tool for TE, as decellularization allows structural preservation of tissue extracellular matrix (ECM) and conservation of its unique cytokine reservoir and the ability to support angiogenesis, cell viability, and proliferation. This represents a major advantage compared to synthetic scaffolds, which can acquire these features only after modification and show limited biocompatibility. In this work, we describe the ability of a skeletal muscle acellular scaffold to promote vascularization both ex vivo and in vivo. Specifically, chicken chorioallantoic membrane assay and protein array confirmed the presence of pro-angiogenic molecules in the decellularized tissue such as HGF, VEGF, and SDF-1α. The acellular muscle was implanted in BL6/J mice both subcutaneously and ortotopically. In the first condition, the ECM-derived scaffold appeared vascularized 7 days post-implantation. When the decellularized diaphragm was ortotopically applied, newly formed blood vessels containing CD31⁺, αSMA⁺, and vWF⁺ cells were visible inside the scaffold. Systemic injection of Evans Blue proved function and perfusion of the new vessels, underlying a tissue-regenerative activation. On the contrary, the implantation of a synthetic matrix made of polytetrafluoroethylene used as control was only surrounded by vWF⁺ cells, with no cell migration inside the scaffold and clear foreign body reaction (giant cells were visible). The molecular profile and the analysis of macrophages confirmed the tendency of the synthetic scaffold to enhance inflammation instead of regeneration. In conclusion, we identified the angiogenic potential of a skeletal muscle-derived acellular scaffold and the pro-regenerative environment activated in vivo, showing clear evidence that the decellularized diaphragm is a suitable candidate for skeletal muscle tissue engineering and regeneration.

Single-cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers.

BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.

Amniotic fluid stem cells restore the muscle cell niche in a HSA-Cre, Smn(F7/F7) mouse model.

Mutations in the survival of motor neuron gene (SMN1) are responsible for spinal muscular atrophy, a fatal neuromuscular disorder. Mice carrying a homozygous deletion of Smn exon 7 directed to skeletal muscle (HSA-Cre, Smn(F7/F7) mice) present clinical features of human muscular dystrophies for which new therapeutic approaches are highly warranted. Herein we demonstrate that tail vein transplantation of mouse amniotic fluid stem (AFS) cells enhances the muscle strength and improves the survival rate of the affected animals. Second, after cardiotoxin injury of the Tibialis Anterior, only AFS-transplanted mice efficiently regenerate. Most importantly, secondary transplants of satellite cells (SCs) derived from treated mice show that AFS cells integrate into the muscle stem cell compartment and have long-term muscle regeneration capacity indistinguishable from that of wild-type-derived SC. This is the first study demonstrating the functional and stable integration of AFS cells into the skeletal muscle, highlighting their value as cell source for the treatment of muscular dystrophies.

Microfluidic reprogramming to pluripotency of human somatic cells.

Human induced pluripotent stem cells (hiPSCs) have a number of potential applications in stem cell biology and regenerative medicine, including precision medicine. However, their potential clinical application is hampered by the low efficiency, high costs, and heavy workload of the reprogramming process. Here we describe a protocol to reprogram human somatic cells to hiPSCs with high efficiency in 15 d using microfluidics. We successfully downscaled an 8-d protocol based on daily transfections of mRNA encoding for reprogramming factors and immune evasion proteins. Using this protocol, we obtain hiPSC colonies (up to 160 ± 20 mean ± s.d (n = 48)) in a single 27-mm2 microfluidic chamber) 15 d after seeding ~1,500 cells per independent chamber and under xeno-free defined conditions. Only ~20 µL of medium is required per day. The hiPSC colonies extracted from the microfluidic chamber do not require further stabilization because of the short lifetime of mRNA. The high success rate of reprogramming in microfluidics, under completely defined conditions, enables hundreds of cells to be simultaneously reprogrammed, with an ~100-fold reduction in costs of raw materials compared to those for standard multiwell culture conditions. This system also enables the generation of hiPSCs suitable for clinical translation or further research into the reprogramming process.

Generation of a Functioning and Self-Renewing Diaphragmatic Muscle Construct.

Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off-the-shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three-dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self-renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019;8:858&869.

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

Endothelial properties of third-trimester amniotic fluid stem cells cultured in hypoxia.

INTRODUCTION: Endothelial dysfunction is found in different pathologies such as diabetes and renal and heart diseases, representing one of the major health problems. The reduced vasodilation of impaired endothelium starts a prothrombotic state associated with irregular blood flow. We aimed to explore the potential of amniotic fluid stem (AFS) cells as a source for regenerative medicine in this field; for the first time, we focused on third-trimester amniotic fluid AFS cells and compared them with the already-described AFS cells from the second trimester. METHODS: Cells from the two trimesters were cultured, selected and expanded in normoxia (20 % oxygen) and hypoxia (5 % oxygen). Cells were analysed to compare markers, proliferation rate and differentiation abilities. Endothelial potential was assessed not only in vitro-Matrigel tube formation assay, acetylated human low-density lipoprotein (AcLDL) uptake-but also in vivo (Matrigel plug with cell injection and two animal models). Specifically, for the latter, we used established protocols to assess the involvement of AFS cells in two different mouse models of endothelial dysfunction: (1) a chronic ischemia model with local injection of cells and (2) an electric carotid damage where cells were systemically injected. RESULTS: We isolated and expanded AFS cells from third-trimester amniotic fluid samples by using CD117 as a selection marker. Hypoxia enhanced the proliferation rate, the surface protein pattern was conserved between the trimesters and comparable differentiation was achieved after culture in both normoxia and hypoxia. Notably, the expression of early endothelial transcription factors and AngiomiRs was detected before and after induction. When in vivo, AFS cells from both trimesters expanded in hypoxia were able to rescue the surface blood flow when locally injected in mice after chronic ischemia damage, and importantly AFS cells at term of gestation possessed enhanced ability to fix carotid artery electric damage compared with AFS cells from the second trimester. CONCLUSIONS: To the best of our knowledge, this is the first research work that fully characterizes AFS cells from the third trimester for regenerative medicine purposes. The results highlight how AFS cells, in particular at term of gestation and cultured in hypoxia, can be considered a promising source of stem cells possessing significant endothelial regenerative potential.