Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.     

Neural crest development is regulated by the transcription factor Sox9

The neural crest is a transient migratory population of stem cells derived from the dorsal neural folds at the border between neural and non-neural ectoderm. Following induction, prospective neural crest cells are segregated within the neuroepithelium and then delaminate from the neural tube and migrate into the periphery, where they generate multiple differentiated cell types. The intrinsic determinants that direct this process are not well defined. Group E Sox genes (Sox8, Sox9 and Sox10) are expressed in the prospective neural crest and Sox9 expression precedes expression of premigratory neural crest markers. Here, we show that group E Sox genes act at two distinct steps in neural crest differentiation. Forced expression of Sox9 promotes neural-crest-like properties in neural tube progenitors at the expense of central nervous system neuronal differentiation. Subsequently, in migratory neural crest cells, SoxE gene expression biases cells towards glial cell and melanocyte fate, and away from neuronal lineages. Although SoxE genes are sufficient to initiate neural crest development they do not efficiently induce the delamination of ectopic neural crest cells from the neural tube consistent with the idea that this event is independently controlled. Together, these data identify a role for group E Sox genes in the initiation of neural crest development and later SoxE genes influence the differentiation pathway adopted by migrating neural crest cells.     

Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

Recent studies have shown that adipose-derived stromal/stem cells (ASCs) contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs). This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2) and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta). NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.     

Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors

In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation.In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.     

Identification and characterisation of the early differentiating cells in neural differentiation of human embryonic stem cells

One of the challenges in studying early differentiation of human embryonic stem cells (hESCs) is being able to discriminate the initial differentiated cells from the original pluripotent stem cells and their committed progenies. It remains unclear how a pluripotent stem cell becomes a lineage-specific cell type during early development, and how, or if, pluripotent genes, such as Oct4 and Sox2, play a role in this transition. Here, by studying the dynamic changes in the expression of embryonic surface antigens, we identified the sequential loss of Tra-1-81 and SSEA4 during hESC neural differentiation and isolated a transient Tra-1-81(-)/SSEA4(+) (TR-/S4+) cell population in the early stage of neural differentiation. These cells are distinct from both undifferentiated hESCs and their committed neural progenitor cells (NPCs) in their gene expression profiles and response to extracellular signalling; they co-express both the pluripotent gene Oct4 and the neural marker Pax6. Furthermore, these TR-/S4+ cells are able to produce cells of both neural and non-neural lineages, depending on their environmental cues. Our results demonstrate that expression of the pluripotent factor Oct4 is progressively downregulated and is accompanied by the gradual upregulation of neural genes, whereas the pluripotent factor Sox2 is consistently expressed at high levels, indicating that these pluripotent factors may play different roles in the regulation of neural differentiation. The identification of TR-S4+ cells provides a cell model for further elucidation of the molecular mechanisms underlying hESC neural differentiation.     

Differentiation of Human Pluripotent Stem Cells into Nephron Progenitor Cells in a Serum and Feeder Free System

Objectives

Kidney disease is emerging as a critical medical problem worldwide. Because of limited treatment options for the damaged kidney, stem cell treatment is becoming an alternative therapeutic approach. Of many possible human stem cell sources, pluripotent stem cells are most attractive due to their self-renewal and pluripotent capacity. However, little is known about the derivation of renal lineage cells from human pluripotent stem cells (hPSCs). In this study, we developed a novel protocol for differentiation of nephron progenitor cells (NPCs) from hPSCs in a serum- and feeder-free system.

Materials and Methods

We designed step-wise protocols for differentiation of human pluripotent stem cells toward primitive streak, intermediate mesoderm and NPCs by recapitulating normal nephrogenesis. Expression of key marker genes was examined by RT-PCR, real time RT-PCR and immunocytochemistry. Each experiment was independently performed three times to confirm its reproducibility.

Results

After modification of culture period and concentration of exogenous factors, hPSCs can differentiate into NPCs that markedly express specific marker genes such as SIX2, GDNF, HOXD11, WT1 and CITED1 in addition to OSR1, PAX2, SALL1 and EYA1. Moreover, NPCs possess the potential of bidirectional differentiation into both renal tubular epithelial cells and glomerular podocytes in defined culture conditions. In particular, approximately 70% of SYN-positive cells were obtained from hPSC-derived NPCs after podocytes induction. NPCs can also form in vitro tubule-like structures in three dimensional culture systems.

Conclusions

Our novel protocol for hPSCs differentiation into NPCs can be useful for producing alternative sources of cell replacement therapy and disease modeling for human kidney diseases.     

Physiological Normoxia and Absence of EGF Is Required for the Long-Term Propagation of Anterior Neural Precursors from Human Pluripotent Cells

Widespread use of human pluripotent stem cells (hPSCs) to study neuronal physiology and function is hindered by the ongoing need for specialist expertise in converting hPSCs to neural precursor cells (NPCs). Here, we describe a new methodology to generate cryo-preservable hPSC-derived NPCs that retain an anterior identity and are propagatable long-term prior to terminal differentiation, thus abrogating regular de novo neuralization. Key to achieving passagable NPCs without loss of identity is the combination of both absence of EGF and propagation in physiological levels (3%) of O₂. NPCs generated in this way display a stable long-term anterior forebrain identity and importantly retain developmental competence to patterning signals. Moreover, compared to NPCs maintained at ambient O₂ (21%), they exhibit enhanced uniformity and speed of functional maturation, yielding both deep and upper layer cortical excitatory neurons. These neurons display multiple attributes including the capability to form functional synapses and undergo activity-dependent gene regulation. The platform described achieves long-term maintenance of anterior neural precursors that can give rise to forebrain neurones in abundance, enabling standardised functional studies of neural stem cell maintenance, lineage choice and neuronal functional maturation for neurodevelopmental research and disease-modelling.     

Neural induction of porcine‐induced pluripotent stem cells and further differentiation using glioblastoma‐cultured medium

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine‐induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma‐ and Mad‐related protein (SMAD) inhibitors, a TGF‐β inhibitor and BMP4 inhibitor. The dSMADi‐derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid‐treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma‐cultured medium including injury‐related cytokines treated porcine iPSC‐NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.     

New bioengineering insights into human neural precursor cell expansion in culture

Understanding initial cell growth, interactions associated with the process of expansion of human neural precursor cells (hNPCs), and cellular events pre- and postdifferentiation are important for developing bioprocessing protocols to reproducibly generate multipotent cells that can be used in basic research or the treatment of neurodegenerative disorders. Herein, we report the in vitro responses of telencephalon hNPCs grown in a serum-free growth medium using time-lapse live imaging as well as cell-surface marker, aggregate size, and immunocytochemical analyses. Time-lapse analysis of hNPC initial expansion indicated that cell-surface attachment in stationary culture and the frequency of cell-cell interaction in suspension conditions are important for subsequent aggregate formation and hNPC growth. In the absence of cell-surface attachment in low-attachment stationary culture, large aggregates of cells were formed and expansion was adversely affected. The majority of the telencephalon hNPCs expressed CD29, CD90, and CD44 (cell surface markers involved in cell-ECM and cell-cell interactions to regulate biological functions such as proliferation), suggesting that cell-surface attachment and cell-cell interactions play a significant role in the subsequent formation of cell aggregates and the expansion of hNPCs. Before differentiation, about 90% of the cells stained positive for nestin and expressed two neural precursor cells surface markers (CD133 and CD24). Upon withdrawal of growth cytokines, hNPCs first underwent cell division and then differentiated preferentially towards a neuronal rather than a glial phenotype. This study provides key information regarding human NPC behavior under different culture conditions and favorable culture conditions that are important in establishing reproducible hNPC expansion protocols.     

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.     

Neural and oligodendrocyte progenitor cells: transferrin effects on cell proliferation

NSC (neural stem cells)/NPC (neural progenitor cells) are multipotent and self-renew throughout adulthood in the SVZ (subventricular zone) of the mammalian CNS (central nervous system). These cells are considered interesting targets for CNS neurodegenerative disorder cell therapies, and understanding their behaviour in vitro is crucial if they are to be cultured prior to transplantation. We cultured the SVZ tissue belonging to newborn rats under the form of NS (neurospheres) to evaluate the effects of Tf (transferrin) on cell proliferation. The NS were heterogeneous in terms of the NSC/NPC markers GFAP (glial fibrillary acidic protein), Nestin and Sox2 and the OL (oligodendrocyte) progenitor markers NG2 (nerve/glia antigen 2) and PDGFRα (platelet-derived growth factor receptor α). The results of this study indicate that aTf (apoTransferrin) is able to increase cell proliferation of SVZ-derived cells in vitro, and that these effects were mediated at least in part by the TfRc1 (Tf receptor 1). Since OPCs (oligodendrocyte progenitor cells) represent a significant proportion of the proliferating cells in the SVZ-derived primary cultures, we used the immature OL cell line N20.1 to show that Tf was able to augment the proliferation rate of OPC, either by adding aTf to the culture medium or by overexpressing rat Tf in situ. The culture medium supplemented with ferric iron, together with aTf, increased the DNA content, while ferrous iron did not. The present work provides data that could have a potential application in human cell replacement therapies for neurodegenerative disease and/or CNS injury that require the use of in vitro amplified NPCs.     

The effect of cell synchronization upon the detection of T and B lymphoid cell receptors on two continuous lymphoid cell lines.

In the present study, the effect of the cell synchronization on the detection of T and B cell surface markers of two continuous lines of lymphoid cells (FL-74 and CT45-S) was examined. Suspension cultures were synchronized by deprivation of isoleucine and surface markers were quantitated by T rosette formation with guinea pig erythrocytes (E) and B rosette formation with an erythrocyte-antibody-complement (EAC) complex. After 24 hr, cells were resuspended in complete culture medium. Virtually 100% of FL-74 cells expressed the T cell marker at time 0, with a progressive decline to 80% at saturation density. A bell-shaped curve for expression of the EAC marker on CT45-S cells was seen with maximum expression in the logarithmic phase of the growth cycle. Spent culture medium was examined for the presence of free soluble receptor. Preincubation of E and EAC in appropriate old medium resulted in 42% inhibition of E rosettes and 42% inhibition of EAC rosettes with FL-74 and CT45-S cells, respectively. Thus quantitation of lymphocyte subpopulations as B, T or null cells with these cellular markers may be influenced by the age of the cell examined, phase of the cell cycle and the amount of free receptor present in the surrounding medium.     

Sharpening of the anterior neural border in the chick by rostral endoderm signalling

The anterior border of the neural plate, presumed to contain the prospective peripheral portion (roof) of the prospective telencephalon, emerges within a vaguely defined proneural ectodermal region. Fate maps carried out at HH4 in the chick reveal that this region still produces indistinctly neural, placodal and non-neural derivatives; it does not express neural markers. We examined how the definitive anterior border domain of the rostral forebrain becomes established and comes to display a neural molecular profile, whereas local non-neural derivatives become separated. The process, interpreted as a border sharpening mechanism via intercalatory cell movements, was studied using fate mapping, time-lapse microscopy and in situ hybridization. Separation of neural and non-neural domains proceeds along stages HH4-HH4+, is well advanced at HH5, and is accompanied by a novel dorsoventral intercalation, oriented orthogonal to the border, that distributes transitional cells into molecularly distinct neural and non-neural fields. Meanwhile, neuroectodermal Sox2 expression spreads peripherally from the neighbourhood of the node, reaching the nascent anterior border domain at HH5. We also show that concurrent signals from the endodermal layer are necessary to position and sharpen the neural border, and suggest that FGF8 might be a component of this signalling.     

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell‐Induced Photoreceptor Survival

Retinal degenerative diseases lead to blindness with few treatments. Various cell‐based therapies are aimed to slow the progression of vision loss by preserving light‐sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co‐culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co‐cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) mediated oxidative response signaling as an hNPC‐induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.     

The effect of cell synchronization upon the detection of T and B lymphoid cell receptors on two continuous lymphoid cell lines

Summary In the present study, the effect of the cell synchronization on the detection of T and B cell surface markers of two continuous lines of lymphoid cells (FL-74 and CT45-S) was examined. Suspension cultures were synchronized by deprivation of isoleucine and surface markers were quantitated by T rosette formation with guinea pig erythrocytes (E) and B rosette formation with an erythrocyte-antibody-complement (EAC) complex. After 24 hr, cells were resuspended in complete culture medium. Virtually 100% of FL-74 cells expressed the T cell marker at time 0, with a progressive decline to 80% at saturation density. A bell-shaped curve for expression of the EAC marker on CT45-S cells was seen with maximum expression in the logarithmic phase of the growth cycle. Spent culture medium was examined for the presence of free soluble receptor. Preincubation of E and EAC in appropriate old medium resulted in 42% inhibition of E rosettes and 42% inhibition of EAC rosettes with FL-74 and CT45-S cells, respectively. Thus quantitation of lymphocyte subpopulations as B, T or null cells with these cellular markers may be influenced by the age of the cell examined, phase of the cell cycle and the amount of free receptor present in the surrounding medium. This research was supported in part by contract NO1 CP 5-3571 with the Virus Cancer Program of the NCI, NIH, PHS grant no. 2 RO1 A1-09022-07, Allergy and Infectious Diseases NIH, PHS and The State of Ohio Canine Research Funds.     

The Olfactory Bulb in Newborn Piglet Is a Reservoir of Neural Stem and Progenitor Cells

The olfactory bulb (OB) periventricular zone is an extension of the forebrain subventricular zone (SVZ) and thus is a source of neuroprogenitor cells and neural stem cells. While considerable information is available on the SVZ-OB neural stem cell (NSC)/neuroprogenitor cell (NPC) niche in rodents, less work has been done on this system in large animals. The newborn piglet is used as a preclinical translational model of neonatal hypoxic-ischemic brain damage, but information about the endogenous sources of NSCs/NPCs in piglet is needed to implement endogenous or autologous cell-based therapies in this model. We characterized NSC/NPC niches in piglet forebrain and OB-SVZ using western blotting, histological, and cell culture methods. Immunoblotting revealed nestin, a NSC/NPC marker, in forebrain-SVZ and OB-SVZ in newborn piglet. Several progenitor or newborn neuron markers, including Dlx2, musashi, doublecortin, and polysialated neural cell adhesion molecule were also detected in OB-SVZ by immunoblotting. Immunohistochemistry confirmed the presence of nestin, musashi, and doublecortin in forebrain-SVZ and OB-SVZ. Bromodeoxyuridine (BrdU) labeling showed that the forebrain-SVZ and OB-SVZ accumulate newly replicated cells. BrdU-positive cells were immunolabeled for astroglial, oligodendroglial, and neuronal markers. A lateral migratory pathway for newly born neuron migration to primary olfactory cortex was revealed by BrdU labeling and co-labeling for doublecortin and class III β tubulin. Isolated and cultured forebrain-SVZ and OB-SVZ cells from newborn piglet had the capacity to generate numerous neurospheres. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Neurosphere-derived cells differentiated into neurons, astrocytes, and oligodendrocytes and were amenable to permanent genetic tagging with lentivirus encoding green fluorescent protein. We conclude that the piglet OB-SVZ is a reservoir of NSCs and NPCs suitable to use in autologous cell therapy in preclinical models of neonatal/pediatric brain injury.