In vitro modelling of cortical neurogenesis by sequential induction of human umbilical cord blood stem cells.

Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.

The simplest method for in vitro ß-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), Wharton's 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.

The cord blood separation league table: a comparison of the major clinical grade harvesting techniques for cord blood stem cells.

BACKGROUND AND OBJECTIVES: Well over 1 million Umbilical Cord Blood units (UCB) have been stored globally in the last 10 years. Already, over 20,000 transplants been performed using UCB for haematopoietic reconstitution alone, now this potential is joined by regenerative medicine. However, more needs to be known about processing of this stem cell source for it to reach full potential. METHODS AND RESULTS: IN THIS STUDY WE EVALUATED FIVE SEPARATION METHODS: plasma depletion, density gradient, Hetastarch, a novel method known as PrepaCyte-CB and an automated centrifugal machine. Sepax gives the highest recovery of nucleated cells, an average of 78.8% (SD±21.36). When looking at CD34+ haematopoietic stem cells PrepaCyte-CB provided the greatest recovery at 74.47% (SD±8.89). For volume reduction density gradient was the most effective leaving 0.03×10(6) RBC/ml, 8 times more efficient than its nearest competitor PrepaCyte-CB (p<0.05). Finally PrepaCyte-CB processing left samples with the highest clonogenic potential after processing and more significantly after cryopreservation: 9.23 CFU/10(8) cells (SD±2.33), 1.5 fold more effective than its nearest rival Sepax (p<0.05). CONCLUSIONS: PrepaCyte-CB was the most flexible method; the only processing type unaffected by volume. Results indicate that processing choice is important depending on your final intended use.

Umbilical cord blood stem cells--an ethical source for regenerative medicine.

Never before in the history of human kind have we tried to keep a tissue preserved for the entire life of a human. This, however, is what is proposed with umbilical cord blood banking. The media often report on the latest 'Stem Cell' breakthrough in 'Regenerative Medicine'. Between human embryonic stem cells, adult stem cells and umbilical cord blood stem cells, it is hard to decipher what all these have done and will do for patients. Cord blood is collected after birth with no harm to mother or baby and contains stem cells with an incredible potential to form tissues including neural, liver and pancreatic tissues in a laboratory. In less than 20 years, cord blood has become the biggest real success story in stem cells, with nearly 10,000 patients treated, from blood and immune transplants, through to more controversial interventions, such as Type 1 diabetes. With ongoing clinical trials and research predicting new avenues, the future seems assured for this stem cell source. What does this predict, however, for cord blood banking? More importantly, what does it justify?

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 microm 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.

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.

Embryonic-like stem cells from umbilical cord blood and potential for neural modeling.

Stem cells offer the distinct prospect of changing the face of human medicine. However, although they have potential to form different tissues, are still in the early stages of development as therapeutic interventions. The three most used stem cell sources are umbilical cord blood, bone marrow and human embryos. Whilst, cord blood is now used to treat over 70 disorders, at the time of writing this manuscript, not a single disease has been overcome or ameliorated using human embryonic stem cells. Advancing stem cell medicine requires ethically sound and scientifically robust models to develop tomorrow's medicines. Media attention, however, distracts from this reality; it is important to remember that stem cells are a new visitor to the medical world and require more research. Here we describe the utility of human cord blood to develop neural models that are necessary to take stem cells to the next level--into human therapies.

Umbilical cord blood stem cells can expand hematopoietic and neuroglial progenitors in vitro.

The ability of hematopoietic tissue-derived adult stem cells to transdifferentiate into neural progenitor cells offers an interesting alternative to central nervous system (CNS)- or embryonic-derived stem cells as a viable source for cellular therapies applied to brain regeneration. Umbilical cord blood (CB) due to its primitive nature and it unproblematic collection appears as a promising candidate for multipotent stem cell harvest. We developed a negative immunomagnetic selection method that depletes CB from hematopoietic lineage marker-expressing cells, hence isolating a discrete lineage negative (LinNeg) stem cell population (0.1% of CB mononucleated cell [MCN] population). In liquid culture supplemented with thrombopoietin, flt-3 ligand, and c-kit ligand (TPOFLK), CB LinNeg stem cells could expand primitive nonadherent hematopoietic progenitors (up to 47-fold) and simultaneously produce slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks. Laser scanning confocal microscopy analysis identified these adherent cells to express glial fibrillary acidic protein (GFAP). Gene expression analysis showed upregulation of primitive neuroglial progenitor cell markers including, GFAP, nestin, musashi-1, and necdin. ELISA quantification of liquid culture supernatant revealed the in vitro release of transforming growth factor beta-1 (TGFbeta1), glial cell line-derived neurotrophic factor (GDNF) suggesting their contribution to CB LinNeg stem cell transdifferentiation into neuroglial progenitors. Our study supports that a single CB specimen can be pre-expanded in TPOFLK to produce both primitive hematopoietic and neuropoietic progenitors, hence widening CB clinical potential for cellular therapies.

Characterization of a lineage-negative stem-progenitor cell population optimized for ex vivo expansion and enriched for LTC-IC.

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34+ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133+ cells to represent a more primitive cell population than their CD34+ counterparts. However, both CD34+ and CD133+ cells still encompass cells at various stages of maturation, possibly impairing long-term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long-term in vivo hematopoiesis. We report here an optimized, rapid negative-isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage-negative (Lin-) cell population (0.10% +/- 0.02% MNC, n=12). This primitive Lin- cell population encompassed CD34+/- and CD133+/- HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin- cells being highly enriched for long-term culture-initiating cells when compared with both CD133+ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c-Kit ligand 20 ng/ml) Lin- cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133+ cells. Therefore, our Lin- stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well-characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.

Multiparametric analysis of immature cell populations in umbilical cord blood and bone marrow.

Adult stem cells are finding increased therapeutic potential not least in tissue regeneration protocols. The cell sources being proposed for such protocols include embryonic, umbilical cord blood (CB) and adult bone marrow (BM). Although embryonic sources are controversial, CB and marrow are available immediately. The appropriate cells of use in these sources are considered to be extremely rare and a characterisation of the starting cell source is important for the development of adult stem cell protocols and ex vivo expansion. Umbilical CB and BM mononuclear cells were labelled for the antigens CD34, CD133, CD117, CD164, Thy-1 or CD38, and additional intracellular CD34 antigen. Three dimensional flow-cytometric analyses were carried out together with dual laser confocal microscope analysis for antigen profile expression. Variable levels of immaturity were detected on CB and BM populations using internal and external CD34 antigen. For CB and BM cells, internal CD34 (intCD34+) could be detected on co-expressing CD133+ cells before expression of external CD34 antigen (extCD34+). CD38 co-expression analysis also showed that a small but distinct group of cells expressing low CD38 and no external CD34 antigen could be detected. Additional phenotyping of these cells using CD117, Thy-1, CD164 and CD133 demonstrated variable primitive status detectable within the external CD34- population. Newly harvested primary CB and BM populations were shown to contain not only cellular populations of known standard sequential maturity but also populations of more extreme rarity. The presence of cells which lacked extracellular CD34 antigen, in both CB and BM, but which possessed CD133, has important implications for purification of human stem cells in clinical applications.

Colocalization analysis of sialomucins CD34 and CD164.

Flow cytometric protocols are employed to identify and characterize hemopoietic stem/progenitor populations before transplantation. Cell surface antigens, including CD34, are employed in this process and widely used in harvest protocols, which largely ignores the potential functional role of such antigens. Transmembrane glycoprotein sialomucins, including CD34 and CD164, have been implicated in cell-to-cell interactions and activation. CD164, also expressed on early hemopoietic populations, was reported to have a possible function facilitating CD34(+) cells to adhere to bone marrow stroma. In this study, we employed high-definition laser-scanning confocal microscopy to investigate CD34 and CD164 surface co-localization patterns on bone marrow and cord blood cells and to compare the expression patterns using a three-dimensional computer-generated method developed in house. Differential interference microscopy analysis revealed bone marrow membrane activity was higher than the corresponding cord blood counterpart, perhaps indicating the marrow microenvironmental nature. Fluorescence analysis of CD34 and CD164 antigens showed both were expressed first in a halo-like pattern and second in antigen-dense pockets. Three-dimensional computer analyses further revealed that this pocketing corresponded to dense crest-like surface structures appearing to rise from the point of adherence on the slide. Further, it was found that CD34 and CD164 display strong colocalization patterns on cells expressing both antigens. The dual nature of the CD34 and CD164 antigens discovered here lends further evidence to the previous literature implicating a strong functional link between these two sialomucins, which should be considered in the transplantation arena and in the function of such sialomucins as negative regulators of cell proliferation.

Diaminofluorene stain detects erythroid differentiation in immature haemopoietic cells treated with EPO, IL-3, SCF, TGFbeta1, MIP-1alpha and IFNgamma.

We have combined in vitro clonogenic culture and a highly sensitive stain for haemoglobin to compare the influence of EPO, IL-3, SCF, TGFbeta1, MIP-1alpha and IFNgamma, to directly stimulate cells in the progenitor compartment to develop towards the erythroid lineage. Three cell lines were chosen, as they exist developmentally arrested in the progenitor compartment, yet in a pliant state of maturation. HEL (erythroleukaemia) and K562 (CML-derived) cell lines, may, under appropriate stimuli, develop erythroid characters, whilst the third, U937 (as control cell line), may be stimulated by DMSO to differentiate to myeloid cells. After in vitro semi-solid methylcellulose culture with these cytokines, resulting colonies were stained with 2,7-diaminofluorene (DAF), which sensitively stains haemoglobin blue. Haemoglobin production was low in HEL and K562 cells and absent in U937. Cytokine analysis showed varying levels of influence depending on the starting level of cell line maturation. EPO and TGFbeta1 maximally stimulated haemoglobin production in the HEL and K562 cell lines. This differential cytokine stimulation analysis combined with sensitive DAF haemoglobin detection could be applied in the study of many erythropoiesis-deficient patients or primitive erythropoiesis.

AC133+ umbilical cord blood progenitors demonstrate rapid self-renewal and low apoptosis.

Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133+ cells (2-4 x 10(4)/ml), mononuclear cells (MNC, 1-2 x 10(6)/ml) and AC133negative cells (AC133(neg), 2-4 x 10(4)/ml) in stroma-free 8 d liquid culture (fetal bovine serum-supplemented Iscove's-modified Dulbecco's medium (IMDM) with either 'K36EG'[c-Kit ligand, interleukin 3 (IL-3), IL-6, erythropoietin, granulocyte colony-stimulating factor] or 'TPOFL' (thrombopoietin, Flt-3 ligand). Cell enumeration, apoptosis assay and AC133/CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133+ cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133(neg) populations (K36EG FI: 15.04 +/- 5.46; TPOFL FI: 8.59 +/- 2.92, P < 0.05). After 8 d, populations lacking AC133+ cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0.01). Also, when compared with K36EG, 8 d TPOFL-expanded AC133+ cells encompassed a significantly higher percentage of AC133+ and CD34+ early HSPC (K36EG: 20.50 +/- 2.36; TPOFL: 47.00 +/- 7.69; P < 0.05). In conclusion, TPOFL synergism demonstrated the potential for AC133+ HSPC ex vivo expansion inducing self-renewal, early HSPC maintenance and promoting cell survival status.