Neural stem cell transplants improve cognitive function without altering amyloid pathology in an APP/PS1 double transgenic model of Alzheimer's disease.

Neural stem cells (NSCs) are capable of self-renewal and are multipotent. Transplantation of NSCs may represent a promising approach for treating neurodegenerative disorders associated with cognitive decline, such as Alzheimer disease (AD) characterized by extensive loss of neurons. In this study, we investigated the effect of NSC transplantation on cognitive function in the amyloid precursor protein/presenilin-1 (APP/PS1) transgenic mouse, an AD mouse model with age-dependent cognitive deficits. We found that NSCs bilaterally transplanted into hippocampal regions improved spatial learning and memory function in these mice, but did not alter Aß pathology. Immunohistochemical analyses determined that NSCs proliferated, migrated, and differentiated into three neuronal cell types. The improvement in cognitive function was correlated with enhanced long-term potentiation (LTP) and an increase in the neuron expression of proteins related to cognitive function: N-methyl-D-aspartate (NMDA) 2B unit, synaptophysin (SYP), protein kinase C ζ subtypes (PKCζ), tyrosine receptor kinase B (TrkB), and brain-derived neurotrophic factor (BDNF). Taken together, our data indicated that injected NSCs can rescue cognitive deficits in APP/PS1 transgenic mice by replacing neuronal cell types expressing multiple cognition-related proteins that enhance LTP.

MicroRNAs in regulation of pluripotency and somatic cell reprogramming: small molecule with big impact.

MicroRNAs (miRNAs), a group of small non-coding RNAs, have emerged as significant modulators in the establishment and generation of pluripotency, a developmental process that consists of complex cell-fate arrangements. The finding of embryonic stem cell (ESC) cycle-specific miRNAs reveals an important regulation scheme of pluripotency. Subsequent studies showed the ESC-enriched or ESC-depleted miRNAs can regulate induced pluripotent stem cells(iPSC). Moreover, miRNA profiling of iPSC and ESC may distinguish them from one another and facilitate the complex of regulatory network. The accumulative effects of miRNA action enable using miRNA alone to generate iPSCs. Despite the robustness of iPSC studies, further investigations are needed since miRNA may have more impact on induced pluripotency, and the roles of miRNAs in somatic cell nuclear transfer (SCNT), another approach toward cellular reprogramming, remains unclear. This point-of-view article will discuss miRNAs and their impact on the normal and induced pluripotency, as well as bring new insights on somatic cell reprogramming.

Detection of neural stem cells function in rats with traumatic brain injury by manganese-enhanced magnetic resonance imaging.

BACKGROUND: Previously we had successfully tracked adult human neural stem cells (NSCs) labeled with superparamagnetic iron oxide particles (SPIOs) in host human brain after transplantation in vivo non-invasively by magnetic resonance imaging (MRI). However, the function of the transplanted NSCs could not be evaluated by the method. In the study, we applied manganese-enhanced MRI (ME-MRI) to detect NSCs function after implantation in brain of rats with traumatic brain injury (TBI) in vivo. METHODS: Totally 40 TBI rats were randomly divided into 4 groups with 10 rats in each group. In group 1, the TBI rats did not receive NSCs transplantation. MnCl2·4H2O was intravenously injected, hyperosmolar mannitol was delivered to disrupt rightside blood brain barrier, and its contralateral forepaw was electrically stimulated. In group 2, the TBI rats received NSCs (labeled with SPIO) transplantation, and the ME-MRI procedure was same to group 1. In group 3, the TBI rats received NSCs (labeled with SPIO) transplantation, and the ME-MRI procedure was same to group 1, but diltiazem was introduced during the electrical stimulation period. In group 4, the TBI rats received phosphate buffered saline (PBS) injection, and the ME-MRI procedure was same to group 1. RESULTS: Hyperintense signals were detected by ME-MRI in the cortex areas associated with somatosensory in TBI rats of group 2. These signals, which could not be induced in TBI rats of groups 1 and 4, disappeared when diltiazem was introduced in TBI rats of group 3. CONCLUSION: In this initial study, we mapped implanted NSCs activity and its functional participation within local brain area in TBI rats by ME-MRI technique, paving the way for further pre-clinical research.

Fates of donor and recipient mitochondrial DNA during generation of interspecies SCNT-derived human ES-like cells.

To investigate nuclear donor and cytoplast recipient mitochondria fate and their effects on generation of interspecies somatic cell nuclear transfer (iSCNT)-derived human embryonic stem (ES)-like cells, iSCNT embryos were reconstructed between enucleated goat oocytes and human neural stem cells (hNSCs). A total of 10.74% cleaved embryos (13/121) developed to blastocyst stage. One typical primary ES-like (tpES-like) colony and two nontypical primary ES-like (non-tpES-like) colonies designated as non-tpES-like cell-1 and non-tpES-like cell-2, respectively, were obtained from the inner cell masses of iSCNT blastocysts. The tpES-like cells expressed ESC markers. Both human and goat mtDNA could be detected in the embryos at 2-8-, 16-32-cell, and blastocyst stages, and in tpES-like colony and two non-tpES-like colonies. Human mtDNA copies per cell from embryos at two- to eight-cell stage to the three colonies maintain almost its original level, whereas 2.88 x 10(5) goat mtDNA copies per oocyte decreased to 10.8 copies per tpES-like cell, 493 copies per non-tpES-like cell-1, and 77.6 copies per non-tpES-like cell-2, resulting in 43.75% (8.4/19.2), 1.24% (6.2/499), and 14.63% (13.3/90.9) mtDNA content in tpES-like cell, non-tpES-like cell-1, and non-tpES-like cell-2 was that of nuclear donor, respectively. Human-specific Tfam and Polg mRNA could be detected in cells of the three colonies. However, tpES-like colony failed to be passaged. The mRNA level of CoxIV encoded by nuclear donor in tpES-like cell was higher than that in non-tpES-like cell, but significantly lower than that of human ESC, suggesting proper nuclear-cytoplasmic communication would not be established in tpES-like cells. Thus, the data suggest that (1) goat oocytes could reprogram human neural stem cells (hNSCs) into embryonic state and further support the inner cell mass (ICM) of iSCNT blastocyst to form tpES-like colony; (2) nuclear donor mtDNA could be replicated and maintain its original level during the reduction of recipient mitochondrial DNA copies, (3) nuclear-cytoplasmic communication and recipient mtDNA copies might affect the derivation of iSCNT-derived ES-like cells.

Different intervals of ovum pick-up affect the competence of oocytes to support the preimplantation development of cloned bovine embryos.

The objective of this study was to determine the effect of different frequencies of transvaginal ovum pick-up (OPU) on the quantity of recovered cumulus oocyte complexes (COCs) and subsequently the competence of matured oocytes to support the preimplantation development of cloned bovine embryos. The COCs were aspirated from the ovaries of 6 Chinese Holstein cows by transvaginal follicle aspiration twice a week (every 3 or 4 days) (Group I), every 5 days (Group II), once a week (every 7 days) (Group III), every 10 days (Group IV), and once every 2 weeks (every 14 days) (Group V). The developmental stages of the follicles were confirmed by the diameter of the dominant follicle (DF) and harvested COCs, and the dynamics of the follicular wave were clarified. In addition, extrusions of the first polar body (PB I) from the oocytes were observed at different time intervals after the initiation of in vitro maturation (IVM) to identify the appropriate culture time window for somatic cell nuclear transfer. Matured oocytes were used to produce cloned bovine embryos that were subsequently cultured in the goat oviduct. After 7 days, the embryos were flushed out, and the developmental rates of the blastocysts were compared among the five groups. The results showed that the aspirations of all follicles >or=3 mm in diameter (D1) induced and synchronized the dynamics of the follicular wave, and the subordinate follicles became atretic after 4 days (D5). Another follicular wave started between D7 and D10, and atresia in the subordinate follicles in the second follicular wave began on D14. The timing of meiotic progression (from the initiation of IVM to the extrusion of PB I) in the oocytes obtained by OPU was later than that of the oocytes obtained from the abattoir. Between 20 and 24 hr after the initiation of IVM, 20% of the oocytes extruded their PB I. Further, 80% (520/650) of the harvested COCs were arrested at metaphase II (MII) by 22 hr of the initiation of IVM and were used as cytoplast donors. The rates of development of the reconstituted embryos to the blastocyst stage were 23.1% (Group I), 15.0% (Group II), 10.9% (Group III), 4.9% (Group IV), and 29.0% (Group V). The results indicate that the developmental potential of follicles from the same living donors were different when different intervals of OPU were adopted and early atretic follicles from the second follicular wave had higher competence to support the early development of cloned bovine embryos.

Development of oocytes from synchronized follicles of early prepubertal goats under exotic hormone control.

The aim of this study was to establish whether early prepubertal Sannen dairy goat could provide a large source of ova for SCNT. The effects of different hormonal treatments including untreated control, FSH alone, estadiol plus progesterone (E2-P4), and E2-P4 and FSH (E2-P4-FSH) on ovary size, follicle number and size were studied using the early prepubertal goats aged 39-60 days. Then prepubertal goats aged 39-120 days were categorized into three groups to study the effect of age on recruited follicle number. The meiotic competence of oocytes derived from > or =3 mm follicles recovered from the early prepubertal goats treated with E2-P4-FSH was compared with those treated with FSH alone. Finally, the development competence of the ova from the early goats treated with E2-P4-FSH was evaluated by SCNT. The E2-P4-FSH treatment produced the largest ovaries, the highest numbers of total follicles and follicles > or =3 mm diameter compared with the other treatments. The number of > or =3 mm follicles per goat treated with E2-P4-FSH was significantly higher for those in the age groups 39-60 days than those in the age groups 61-90 days and 91-120 days. The FSH alone treatment resulted in a lower proportion matured ova in vitro within 27 hr than from those goats treated with E2-P4-FSH. Ova derived from the early prepubetal goats resulted in lower rate of blastocyst in SCNT (15.3% versus 22.1%, P < 0.01) than that of adult goat However, the number of ova recovered per goat was substantially greater for the prepubertal goats (108 +/- 10.3 Versus 28 +/- 5.0). Consequently, the younger goats produced significantly more blastocyst (7.1 +/- 2.7 versus 4.2 +/- 1.4) per head. It was concluded that early pubertal goats treated with E2-P4-FSH could provide a relatively high number of developmentally competent ova for SCNT studies.

[Knockout serum replacement improves establishment efficiency of C57BL/6J mouse embryonic stem cell line].

AIM: To eliminate the influence of serum on self-renewal of embryonic stem cells (ESCs), knockout serum replacement (KSR), a defined formulation, was used to replace serum for the establishment of C57BL/6J mouse ESC line. METHODS: C57BL/6J mouse blastocysts collected at 3.5 days post coitum (d.p.c.) were cultured in the medium supplemented with KSR. In control experiment, KSR was substituted by fetal bovine serum (FBS). When ESC line was established, the morphology of ESCs, the expression of alkaline phosphatase and oct-4, and the karyotype and differentiating ability of ESCs were analyzed. RESULTS: 13 blastocysts were cultured in the medium supplemented with KSR and one ESC line (MES-1) was established with a normal and stable XX karyotype after cultured for more than 20 passages, and then the high expression of alkaline phosphatase and oct-4 was detected. When cultured in suspension, MES-1 formed embryoid bodies. When inoculated subcutaneously into nude mice, MES-1 formed teratoma. After injected into ICR mouse blastocysts collected at 3.5 d.p.c., MES-1 incorporated into the inner cell mass of the host blastocyst and contributed to the development of a chimera. In control experiment, no ESC lines were cultured for more than 3 passages. CONCLUSION: KSR can be efficiently used to isolate and culture C57BL/6J mouse ESCs, which can eliminate traditional prescreening of FBS suitable for isolation and culture of ESCs.

Secondary SCNT doubles the pre-implantation development rate of reconstructed interspecies embryos by using cytoplasts of Sannen dairy goat ova.

The aim of this study was to investigate whether ova of Sannen goat could support the pre-implantation development of interspecies embryos constructed through somatic cell nucleus transfer (SCNT) embryos and whether secondary SCNT (SSCNT) could improve the pre-implantation development of those embryos. The primary SCNT (PSCNT) embryos were produced by using Sannen goat ovum cytoplasts as recipients and fibroblast cells, derived from human, rabbit and Boer goat skins, as nucleus donors. The blastomeres of 8 to 16 cells stage of PSCNT embryos were subsequently used as nucleus donor cells and Sannen goat ovum cytoplasts as recipients to evaluate the effect of SSCNT on the pre-implantation development rate of these reconstructed interspecies embryos. Our results indicate that the pre-implantation development rates of SSCNT embryos reconstructed using these three different blastomeres are almost twice of that of corresponding PSCNT embryos (human, 15.8% vs. 7.8%; rabbit, 27.9% vs. 12.5%; Boer goat 55.3% vs. 24.5%; P < 0.05 in all three cases). The time durations that embryos need for the serial events of remodeling and reprogramming to take place vary, depending on the animal species of nucleus donors. These data suggest that remodeling and reprogramming of donor nucleus may be enhanced by prolonged exposure of donor nucleus to maternal cytoplast. We conclude that Sannen goat cytoplast can support the pre-implantation development of embryos constructed with nuclei from various donors, including fibroblasts of human, rabbit and Boer goat; and the somatic nucleus derived from different species requires more time to achieve its reprogramming necessary for pre-implantation development.

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells.

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.