Nicolas Forraz

Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro

This 3-week protocol produces embryonic-like stem cells from human umbilical cord blood (CBEs) for neural differentiation using a three-step system (cell isolation/expansion/differentiation). The CBE isolation produces a highly purified fraction (CD45−, CD33−, CD7−, CD235a−) of small pluripotent stem cells (2–3 μm in diameter) coexpressing embryonic stem cell markers including Oct4 and Sox2. Initial CBE expansion is performed in high density (5–10 millions per ml) in the presence of extracellular matrix proteins and epidermal growth factor. Subsequent neural differentiation of CBEs requires sequential introduction of morphogenes, retinoic acid, brain-derived neurotrophic factor and cyclic AMP. Described methods emphasize defined media and reagents at all stages of the experiment comparable to protocols described for culturing human embryonic stem cells and cells from other somatic stem cell sources. Neural progenitor and cells generated from CBEs may be used for in vitro drug testing and cell-based assays and potentially for clinical transplantation.

Directed engineering of umbilical cord blood stem cells to produce C-peptide and insulin

Abstract.  Objectives: In this study, we investigated the potential of umbilical cord blood stem cell lineages to produce C‐peptide and insulin. Materials and methods: Lineage negative, CD133+ and CD34+ cells were analyzed by flow cytometry to assess expression of cell division antigens. These lineages were expanded in culture and subjected to an established protocol to differentiate mouse embryonic stem cells (ESCs) toward the pancreatic phenotype. Phase contrast and fluorescence immunocytochemistry were used to characterize differentiation markers with particular emphasis on insulin and C‐peptide. Results: All 3 lineages expressed SSEA‐4, a marker previously reported to be restricted to the ESC compartment. Phase contrast microscopy showed all three lineages recapitulated the treatment‐dependent morphological changes of ESCs as well as the temporally restricted expression of nestin and vimentin during differentiation. After engineering, each isolate contained both C‐peptide and insulin, a result also obtained following a much shorter protocol for ESCs. Conclusions: Since C‐peptide can only be derived from de novo synthesis and processing of pre‐proinsulin mRNA and protein, we conclude that these results are the first demonstration that human umbilical cord blood‐derived stem cells can be engineered to engage in de novo synthesis of insulin.

Potential for access to embryonic‐like cells from human umbilical cord blood

Abstract.  All too often media attention clouds the reality that there are many types of stem cell. The embryos, bone marrow and umbilical cord blood (UCB) are the three most used sources. However, despite what it would appear, embryonic stem cells have not been the first to yield life‐saving cures at present. Faster routes to clinical intervention have been using adult stem cells that can be sourced from bone marrow and from cord blood, and that are readily accessible and are more ethically acceptable to the general public. Both these non‐embryonic sources have been able to provide sufficient numbers of cells to allow development of clinical translational protocols. Bone marrow‐derived cells have been used successfully in myocardial infarct therapy where relining by endothelial tissue has allowed limited reperfusion to damaged cardiac tissue. UCB have also demonstrated significant success for around 20 years in haematotransplantation. With a global human population in excess of 6 billion, births thus UCB, remain the largest untouched source of stem cells available every year. UCB also provide a distinct advantage over other adult stem cells due to the length of the telomere and also due protected immunological status of the developing neonatal environment. The total mutation load in the UCB populations is clearly likely to be significant less than in adult tissues.

The simplest method for in vitro β-cell production from human adult stem cells

Diabetes mellitus is a challenging autoimmune disease. Biomedical researchers are currently exploring efficient and effective ways to solve this challenge. The potential of stem cell therapies for treating diabetes represents one of the major focuses of current research on diabetes treatment. Here, we have attempted to differentiate adult stem cells from umbilical cord blood-derived mesenchymal cells (UCB-MSC), Whartons jelly-derived mesenchymal stem cells (WJ-MSC) and amniotic epithelial stem cells (AE-SC) into insulin-producing cells. The serum-free protocol developed in this study resulted in the differentiation of cells into definitive endoderm, pancreatic foregut, pancreatic endoderm and, finally, pancreatic endocrine cells, which expressed the marker genes SOX17, PDX1, NGN3, NKX6.1, INS, GCG, and PPY, respectively. Detection of the expression of the gap junction-related gene connexin-36 (CX36) using RT-PCR provided conclusive evidence for insulin-producing cell differentiation. In addition to this RT-PCR result, insulin and C-peptide protein were detected by immunohistochemistry and ELISA. Glucose stimulation test results showed that significantly greater amounts of C-peptide and insulin were released from differentiated cells than from undifferentiated cells. In conclusion, the methods investigated in this study can be considered an effective and efficient means of obtaining insulin-producing cells from adult stem cells within a week.

Tuning the mechanical and bioresponsive properties of peptide-amphiphile nanofiber networks.

Here we describe peptide amphiphiles (PAs) which can be self-assembled into nanofiber networks using divalent ions. These networks possess several key properties of extracellular matrix (ECM) including cell-adhesive ligands, enzyme-mediated degradation and self-assembly into hierarchical organization. The self-assembly of PAs and growth of nanofibers could be controlled by modifications of the chemical structure of the PA and/or addition of divalent ions. Altering the length of PAs alters the viscoelastic properties and degradation kinetics of nanofiber networks. Neural cells were successfully encapsulated within nanofiber networks by self-assembly of PAs. Cell adhesive ligands containing nanofiber networks supported neural cells growth, and their cellular behaviors depended on the concentration of cell adhesive ligands. Therefore, we have demonstrated that mechanical properties, degradability, and bioactivity of nanofiber networks could be tuned by altering the chemical composition and the length of PAs.

Experimental therapies for repair of the central nervous system: stem cells and tissue engineering.

Several stem cell‐based therapeutic tools are currently being investigated for the regeneration of central nervous system (CNS) injuries. This review focuses on innovative approaches for CNS tissue repair via the use of implantable cellular devices. These devices are supported by biopharmaceuticals and conventional physiotherapy for the restoration of lost neuronal circuits and CNS function. This paper further reviews new and promising tools currently in pre‐clinical and clinical tests for the treatment of CNS diseases where substantial loss of cellular and extracellular components of neural tissue has occurred such as stroke, encephalopathy and traumatic neural injuries. We also discuss selected 3D bioscaffolds co‐cultured with clinically applicable human mesenchymal stem cells. Recent advances in neural tissue engineering and stem cell differentiation methods have shown promise for their clinical application in treating yet incurable CNS deficits. Copyright

Umbilical cord blood processing using Prepacyte-CB increases haematopoietic progenitor cell availability over conventional Hetastarch separation

Background:  Currently the most frequently used method for umbilical cord blood separation in many hospitals across the UK and the rest of the world, where small‐to‐medium amounts of samples are processed, is Hetastarch, a mechanical, starch‐based method, which causes red cell agglutination by rouleaux formation.

Oct‐4A isoform is expressed in human cord blood‐derived CD133 stem cells and differentiated progeny

Objectives: This study aims to establish whether the pluripotent embryonic stem cell marker and nuclear transcription factor Oct‐4A isoform is expressed in human umbilical cord blood CD133 stem cells (CD133 cells) and their differentiated progeny.