Inhibition of VEGF-A expression in hypoxia-exposed fetal retinal microvascular endothelial cells by exosomes derived from human umbilical cord mesenchymal stem cells

Objective:This study aimed to investigate the potential of human umbilical cord mesenchymal stem cell(hucMSC)-derived exosomes(hucMSC-Exos)in inhibiting hypoxia-induced cell hyper proliferation and overexpression of vascular endothelial growth factor A(VEGF-A)in immature human fetal retinal microvascular endothelial cells(hfRMECs).Methods:Exosomes were isolated from hucMSCs using cryogenic ultracentrifugation and characterized through various techniques,including transmission electron microscopy,nanoparticle tracking analysis,bicinchoninic acid assays,and western blotting.The hfRMECs were identified using von Willebrand factor(vWF)co-staining and divided into four groups:a control group cultured under normoxic condition,a hypoxic model group,a hypoxic group treated with low-concentration hucMSC-Exos(75μg/mL)and a hypoxic group treated with high-concentration hucMSC-Exos(100μg/mL).Cell viability and proliferation were assessed using Cell Counting Kit-8(CCK-8)assay and EdU(5-ethynyl-2′-deoxyuridine)assay respectively.Expression levels of VEGF-A were evaluated using RT-PCR,western blotting and immunofluorescence.Results:Hypoxia significantly increased hfRMECs’viability and proliferation by upregulating VEGF-A levels.The administration of hucMSC-Exos effectively reversed this response,with the high-concentration group exhibiting greater efficacy compared to the lowconcentration group.Conclusion:In conclusion,hucMSC-Exos can dose-dependently inhibit hypoxia-induced hyperproliferation and VEGF-A overexpression in immature fetal retinal microvascular endothelial cells.

Synaptic development in the injured spinal cord cavity following co-transplantation of fetal spinal cord cells and autologous activated Schwann cells

Transplantation of activated transgenic Schwann cells or a fetal spinal cord cell suspension has been widely used to treat spinal cord injury. However, little is known regarding the effects of co-transplantation. In the present study, autologous Schwann cells in combination with a fetal spinal cord cell suspension were transplanted into adult Wistar rats with spinal cord injury, and newly generated axonal connections were observed ultrastructurally. Transmission electron microscopic observations showed that the neuroblast first presented cytoplasmic processes, followed by pre- and postsynaptic membranes with low electron density forming a dense projection. The number and types of synaptic vesicles were increased. Synaptic connections developed from single cell body-dendritic synapses into multiple cell body-dendritic and dendrite-dendritic synapses. In addition, the cell organs of the transplanted neuroblast, oligodendroblast and astroblast matured gradually. The blood-brain barrier appeared subsequently. Moreover, neurofilament, histamine, calcitonin-gene-related peptides, and glial fibrillary acidic protein positive fibers were observed in the transplant region. These findings demonstrate that fetal spinal cord cells in the presence of autologous activated Schwann cells can develop into mature synapses in the cavity of injured spinal cords, suggesting the possibility of information exchange through the reconstructed synapse between fetal spinal cord cells and the host.

An appropriate level of autophagy reduces emulsified isoflurane-induced apoptosis in fetal neural stem cells

Autophagy plays essential roles in cell survival.However,the functions and regulation of the autophagy-related proteins Atg5,LC3B,and Beclin 1 during anesthetic-induced developmental neurotoxicity remain unclear.This study aimed to understand the autophagy pathways and mechanisms that affect neurotoxicity,induced by the anesthetic emulsified isoflurane,in rat fetal neural stem cells.Fetal neural stem cells were cultured,in vitro,and neurotoxicity was induced by emulsified isoflurane treatment.The effects of pretreatment with the autophagy inhibitors 3-methyladenine and bafilomycin and the effects of transfection with small interfering RNA against ATG5(siRNA-Atg5)were observed.Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,and apoptosis was assessed using flow cytometry.Ultrastructural changes were analyzed through transmission electron microscopy.The levels of the autophagy-related proteins LC3B,Beclin 1,Atg5,and P62 and the pro-apoptosis-related protein caspase-3 were analyzed using western blot assay.The inhibition of cell proliferation and that of apoptosis rate increased after treatment with emulsified isoflurane.Autophagolysosomes,monolayer membrane formation due to lysosomal degradation,were observed.The autophagy-related proteins LC3B,Beclin 1,Atg5,and P62 and caspase-3 were upregulated.These results confirm that emulsified isoflurane can induce toxicity and autophagy in fetal neural stem cells.Pre-treatment with 3-methyladenine and bafilomycin increased the apoptosis rate in emulsified isoflurane-treated fetal neural stem cells,which indicated that the complete inhibition of autophagy does not alleviate emulsified isoflurane-induced fetal neural stem cell toxicity.Atg5 expression was decreased significantly by siRNA-Atg5 transfection,and cell proliferation was inhibited.These results verify that the Atg5 autophagy pathway can be regulated to maintain appropriate levels of autophagy,which can inhibit the neurotoxicity induced by emulsified isoflurane anesthetic in fetal neural stem cells.

Enrichment of Fetal Nucleated Red Blood Cells by Multi-core Magnetic Composite Particles for Non-invasive Prenatal Diagnosis

A novel kind of multi-core magnetic composite particles, the surfaces of which were respectively mo- dified with goat-anti-mouse IgG and antitransferrin receptor（anti-CD71）, was prepared. The fetal nucleated red blood cells（FNRBCs） in the peripheral blood of a gravida were rapidly and effectively enriched and separated by the mo- dified multi-core magnetic composite particles in an external magnetic field. The obtained FNRBCs were used for the identification of the fetal sex by means of fluorescence in situ hybridization（FISH） technique. The results demonstrate that the multi-core magnetic composite particles meet the requirements for the enrichment and speration of FNRBCs with a low concentration and the accuracy of detetion for the diagnosis of fetal sex reached to 95%. Moreover, the obtained FNRBCs were applied to the non-invasive diagnosis of Down syndrome and chromosome 3p21 was de- tected. The above facts indicate that the novel multi-core magnetic composite particles-based method is simple, relia- ble and cost-effective and has opened up vast vistas for the potential application in clinic non-invasive prenatal diag- nosis.

Are there fetal stem cells in the maternal brain?

Fetal cells can enter maternal blood during pregnancy but whether they can also cross the blood-brain barrier to enter the maternal brain remains poorly understood. Previous results suggest that fetal cells are summoned to repair damage to the mother＇s brain. If this is confirmed, it would open up new and safer avenues of treatment for brain damage caused by strokes and neural diseases. In this study, we aimed to investigate whether a baby＇s stem cells can enter the maternal brain during pregnancy. Deceased patients who had at least one male offspring and no history of abortion and blood transfusion were included in this study. DNA was extracted from brain tissue samples of deceased women using standard phenol-chloroform extraction and ethanol precipitation methods. Genomic DNA was screened by quantitative fluorescent-polymerase chain reaction amplification together with short tandem repeat markers specific to the Y chromosome, and 13, 18, 21 and X. Any foreign DNA residues that could be used to interpret the presence of fetal stem cells in the maternal brain were monitored. Results indicated that fetal stem cells can not cross the blood-brain barrier to enter the maternal brain.

REGULATORY EFFECTS OF HUMAN RECOMBINANT IL-6 ON NATURAL KILLER CELL ACTIVITY OF HUMAN FETAL SPLEENS

In the present study it was proved first that human recombinant interleukin-6(HrIL-6) significantly augmented natural killer(NK) cell activity derived from human fetal spleens against K562 target cells in a 4 hours 51Cr release assay. The enhancement of NK activity with 24 hours preincubation in HrlL-6 was dose-dependent, and significantly higher than that of fresh NK cells and controls cultured with RPMI-1640 medium alone (P<0.001). We also found that IL-6 was able to augment NK activity from different fetal spleens at 20 to 40 weeks of gestation (up to 2.24 to 2.78 times), and no difference of NK activity of fetal splenocytes treated by HrIL-6 was observed between different fetal age (32.3% to 45.4%, P>0.05). Furthermore, IL-6-augmented NK activity of fetal splenocytes was very similar to adult levels (P>0.05). These finding strongly indicated that IL-6 plays an important role in the development of NK cell function during the gestational period, suggesting that IL-6 may be of importance in the regulation of host defense mechanisms against malignancies and viral diseases.

An Eperimental Study on the Transplantation of Human Fetal Brain Cells into Monkey Models of Parkinson's Disease.

Unilateral administration of methly-phenyl-tetrahydropyridine(MPTP) into the common carotid artery

Chondrogenic priming of human fetal synovium-derived stem cells in an adult stem cell matrix microenvironment

Cartilage defects are a challenge to treat clinically due to the avascular nature of cartilage.Low immunogenicity and extensive proliferation and multidifferentiation potential make fetal stem cells a promising source for regenerative medicine.In this study,we aimed to determine whether fetal synovium-derived stem cells(FSDSCs)exhibited replicative senescence and whether expansion on decellularized extracellular matrix(dECM)deposited by adult SDSCs(AECM)promoted FSDSCs’chondrogenic potential.FSDSCs from passage 2 and 9 were compared for chondrogenic potential,using Alcian blue staining for sulfated glycosaminoglycans(GAGs),biochemical analysis for DNA and GAG amounts,and real-time PCR for chondrogenic genes including ACAN and COL2A1.Passage 3 FSDSCs were expanded for one passage on plastic flasks(PL),AECM,or dECM deposited by fetal SDSCs(FECM).During expansion,cell proliferation was evaluated using flow cytometry for proliferation index,stem cell surface markers,and resistance to hydrogen peroxide.During chondrogenic induction,expanded FSDSCs were evaluated for tri-lineage differentiation capacity.We found that cell expansion enhanced FSDSCs’chondrogenic potential at least up to passage 9.Expansion on dECMs promoted FSDSCs’proliferative and survival capacity and adipogenic differentiation but not osteogenic capacity.AECM-primed FSDSCs exhibited an enhanced chondrogenic potential.

INTRACEREBRAL CO-GRAFTING OF SCHW ANN'S CELLS AND FETAL ADRENAL MEDULLA IN THE TREATMENT OF PARKINSON'S DISEASE

Intracerebral co-grafting of Schwann's cells and human fetal adrenal medullary tissue was performed in 10 patients with Parkinson's disease. One to six months after grafting, symptoms were improved significantly for 1 to 3 grade. Among them, 2 patients resumed nearly normal daily activities. Long-term follow-up showed that the symptoms were not improved satisfactorily in some patients. It is considered that careful selection of patients, administration of amantadine, and co-grafting of Schwann's cells which prompts the survival of chromaffin cells are essential to better results.

Differentiated cells derived from fetal neural stem cells improve motor deficits in a rat model of Parkinson's disease

Objective: Parkinson's disease(PD), which is one of the most common neuro‐degenerative disorders, is characterized by the loss of dopamine(DA) neurons in the substantia nigra in the midbrain. Experimental and clinical studies have shown that fetal neural stem cells(NSCs) have therapeutic effects in neurological disorders. The aim of this study was to examine whether cells that were differentiated from NSCs had therapeutic effects in a rat model of PD. Methods: NSCs were isolated from 14‐week‐old embryos and induced to differentiate into neurons, DA neurons, and glial cells, and these cells were characterized by their expression of the following markers: βⅢ‐tubulin and microtubule‐associated protein 2(neurons), tyrosine hydroxylase(DA neurons), and glial fibrillary acidic protein(glial cells). After a 6‐hydroxydopamine(6‐OHDA)‐lesioned rat model of PD was generated, the differentiated cells were transplanted into the striata of the 6‐OHDA‐lesioned PD rats. Results: The motor behaviors of the PD rats were assessed by the number of apomorphine‐induced rotation turns. The results showed that the NSCs differentiated in vitro into neurons and DA neurons with high efficiencies. After transplantation into the striata of the PD rats, the differentiated cells significantly improved the motor deficits of the transplanted PD rats compared to those of the control nontransplanted PD rats by decreasing the apomorphine‐induced turn cycles as early as 4 weeks after transplantation. Immunofluorescence analyses showed that the differentiated DA neurons survived more than 16 weeks. Conclusions: Our results showed that cells that were differentiated from NSCs had therapeutic effects in a rat PD model, which suggests that differentiated cells may be an effective treatment for patients with PD.

Fate determination of fetal Leydig cells

Leydig cell(LC)is one of the most important somatic cell types in testis,which localized in the interstitium between seminiferous tubules.The major function of Leydig cells is to produce steroid hormone,androgens.LC differentiation exhibits a biphasic pattern in rodent testes,which are divided into two different temporal mature populations,fetal Leydig cells(FLCs)and adult Leydig cells(ALCs).FLCs are transiently present in fetal testes and undergo involution or degeneration after birth.FLCs are completely devoid and replaced by ALCs in adult testes.Comparing to ALCs,FLCs display unique morphology,ultrastructure and functions.The origin of FLCs has been debated for many years,but it is still a mystery.Many factors have been reported regulating the specification,proliferation and differentiation of FLCs.FLCs degenerate in a few weeks postnatally,however,the underlying mechanism is still unknown.In this review,we will focus on the fate determination of FLCs,and summarize the resent progress on the morphology,ultrastructure,function,origin and involution of FLCs.

EFFECTS OF DAURISOLINE ON CYTOSOLIC FREE CALCIUMIN FETAL RAT CEREBRAL CELLS

Cytosolic free Ca([Ca]i)was measured in dissociated cerebral cells isolated from fetal rats with the fluorescent indicater fura-2. Increase in[Ca]i occurred rapidly following explsure of the cells to 50 mmol/L KCI,10mol/L Bayk 8644 or 200μmol/L glutamate（Glu）.[Ca]i elevated by

ALP Induction by β－glycerophosphate during the Nonmineralization Phase In Vitro

β-GP influences on rat osteoblast development at the early period of culture i.e , the non-mineralization phase, and changes with the different cell passages were investigated. Alkaline phosphatase (ALP) was chosen as a main object. Northern blot analysis revealed up to two-fold increase in the steady statelevel of ALP mRNA after stimulation of rat osteoblast with 10 mM β-GP- Likewise, 10 mM β-GP induced a 10─30 % increase in ALP activity (P< 0. 01) of early passages (1 to 4), but not of later passages (5 to 6). The β-GP induced increase in ALP activity was totally inhibited by the protein synthesis inhibitor, cycloheximide (50 μM).β- GP stimulation was found to be without effect on cell proliferation measured as 3H-thymidine incorporation. It is concluded that β-GP has no effect on proliferation but induces an increase in both mRNA level and activity of ALP in the non-mineralization phase of cultures of fetal rat calvarial cells , which lasts for several passages but will disappear in older cultures.

Differentiation of Osteoblast in vitro Is Regulated by Progesterone

The regulation of cellular differentiation by progesterone in fetal rat calvarial osteoblasts was investigated. Our results showed that cells cultured in the presence of progesterone had a 7% increase in the alkaline phosphatase activity when compared to untreated cells. The concentration of osteocalcin in the conditioned medium from progesterone treated osteoblasts was 28% higher than that of untreated controls. In addition,administration of progesterone significantly enhanced the number and area of bone nodules. In conclusion, progesterone stimulates the differentiation of fetal rat calvarial osteoblastic cells in vitro

Neurotrophic factors increase tyrosine hydroxylase immunoreactive cell density of fetal mesencephalic cells

Objective To hibernate fetal ventral mesencephalic tegmental (VMT) cells from Spraque Dawley rats E 15 for 5 days at 4℃ in either hibernation media (HM) or in conditioned hibernation media (CHM) supplemented with trophic factors such as epidermal growth factor (EGF 200 ng/ml), basic fibroblast growth factor (bFGF 100 ng/ml), recombinant human brain derived neurotrophic factor (rhBDNF 20 μg/ml), recombinant human glial cell derived neurotrophic factor (rhGDNF 20 μg/ml), fetal calf serum (FCS 9%), or human placental cord serum (HPCS 10%). Methods The percent of cell viability and the density of tyrosine hydroxylase immunoreactive (THir) cells in fetal striatal VMT co culture were determined. Results The viability of fetal striatal cells (0.8± 0.104) was slightly higher than that of fetal VMT cells (0.7±0.072) at 0 time point (F 17,1 =4.677; P= 0.045). After 5 days of hibernation, the viability of fetal VMT cells reduced by 30% (F 7,1 =88.493; P<0.001) in HM. However, THir cell density reduced by more than 90% as compared to the freshly harvested VMT cells (F 7,1 =179.944; P<0.001). CHM with HPCS, bFGF, EGF, BDNF, and GDNF showed higher THir cell density than that of HM or CHM supplemented with FCS (P<0.001). Conclusion Supplementation of appropriate trophic factors for hibernated fetal VMT cells promotes cell viability and the subsequent expression of THir cell density.

Fetal stem cells are effective in the treatment of Grade Ⅰ and Ⅱrespiratory failure in amyotrophic lateral sclerosis and muscular dystrophy

Objectives: To study the effect of fetal stem cell(FSC) therapy on Grade Ⅰ and Ⅱrespiratory failure in patients with amyotrophic lateral sclerosis(ALS) and muscular dystrophy(MD). Methods: A comparative study was conducted on 41 patients with Grade Ⅰ or Ⅱrespiratory failure(RF) resulting from ALS or MD. The patients were divided into 4 groups according to the underlying disease and the degree of RF. Patients underwent combined treatment, including the experimental application of FSC therapy, and were examined before FSC treatment, and 6 months and 12 months after treatment. Results: FSC treatment improved both subjective and objective breathing parameters as early as 6 months post‐treatment. A significant increase in the forced vital capacity(FVC) and forced expiratory volume in 1 second(FEV1) was reported by all patients with grade Ⅰ RF linked to ALS and MD compared to baseline. Patient respiratory improvement was maintained over the next 6 months. Grade Ⅱ RF patients with MD reported a significant improvement in FVC 12 months after treatment. Conclusions: Evidence for respiratory improvement was observed as early as 6 months in all patients after combined treatment including FSC therapy, and this was maintained for a further 6 months after therapy. In MD patients with Grade Ⅱ RF, treatment resulted in a significant FVC and FEV1 increase within 6 months and downgrading to Grade Ⅰ RF within a year after FSC treatment.

Fetal cell microchimerism in the maternal mouse spinal cord

Fetal cell microchimerism refers to the persistence of fetal cells in the maternal tissues following pregnancy. It has been detected in peripheral organs and the brain, but its existence in the spinal cord has not been reported. Our aim was to detect fetal cell microchimerism in the spinal cord of maternal mice. C57BL/6 female mice were crossed with GFP transgenic male mice and sacrificed after their first or third delivery. GFP-positive cells, which were presumably from fetuses whose fathers were GFP transgenic, were detected in the spinal cord by fluorescence microscopy and immunohistochemistry. PCR was also performed to detect GFP DNA, which must come from GFP hemizygous fetuses. We found GFP-positive cells and detectable GFP DNA in most of the maternal spinal cords. Twenty percent （1/5） of the mice that were only pregnant once had detectable fetal cells, while 80% （4/5） of those that were pregnant three times had detectable fetal cells. Some fetal cells, which not only emitted green fluorescence but also expressed NeuN, were detected in the spinal cords from maternal mice. These results indicate that fetal cells migrate into the spinal cord of a maternal mouse during and/or after the gestational period, and the fetal cells may differentiate into neurons in the spinal cord.

Immunohistochemical characterization of hepatic stem cell-related cells in developing human liver

Little is known about the expression characteristics of the various kinds of possible markers in hepatic stem cells(HSCs)and other HSC-related cells in human fetal liver in various developmental stages.It is significant to investigate the immunohistochemical expression for better understanding of the origin,differentiation and migration of HSCs in the developing human liver.H-E staining and immunohistochemical methods were used to observe the expression of hepatic/cholangiocellular differentiation markers(AFP,GST-p,CK7,CK19)and hematopoietic stem cell markers(CD34 and c-kit)in several kinds of HSC-related cells in thirty cases of fetal liver samples(4–35 weeks after pregnancy).AFP expression appears in fetal hepatocytes at four weeks’gestation.It peaks at 16–24 weeks’gestation and decreases gradually afterwards.Finally,weak signals were only found in some ductal plate cells and a few limiting plate cells.GST-p was detected in hepatic cord cells from the sixth week and in the ductal plate cells from the eighth week.Twenty-six weeks later,only some ductal plate cells and a few limiting plate cells show positive signals.CK19 expression peaks during the 6th–11th weeks in hepatic cord cells and decreases gradually afterwards,except for the ductal plates.CK7 expression was limited in the ductal plate cells and bile ducts cells from the 14th week.CD34 and c-kit were detected at the eighth week in some ductal plate cells and a few mononuclear cells in the hepatic cords/mesenchymal tissue of portal areas.After 21 weeks,CD34 and c-kit were found only in ductal plate cells and a few mononuclear cells in the hepatic mesenchymal tissue of portal areas.Fetal hepatocytes at 4–16 weeks’gestation are mainly constituted by HSCs characterized with bi-potential differentiation capacity.At 16 weeks’gestation,most hepatic cord cells begin to differentiate into hepatocytes and abundant HSCs remain in ductal plate(the origin site of Hering canals).It is also indicated that the hematopoietic stem cells may give rise to some HSCs in embryonic liver.These indirectly support the hypothesis about the location and origin of HSCs in“liver valley hypothesis”reported previously.

THE MECHANISM OF CLINICAL EFFECTIVENESS OF HUMAN FETAL LIVER CELL TRANSFUSION

During the development of human fetus, all organs consisting of various types of cells are in rapid growth. The significance of these cells and the chemical components involved, especially those of special functions in medical treatment of certain refractory diseases, have been noticed gradually. The questions as a step further are therefore raised, e.g. what is the mechanism underlying the clinical effectiveness and how to produce the active substances in large amount by biological engineering technique or chemical synthetic method, instead of using the crude preparation directly derived from fetus.

Recent advances in mammalian reproductive biology

Reproductive biology is a uniquely important topic since it is about germ cells, which are central for transmitting genetic information from generation to generation. In this review, we discuss recent advances in mammalian germ cell development,including preimplantation development, fetal germ cell development and postnatal development of oocytes and sperm. We also discuss the etiologies of female and male infertility and describe the emerging technologies for studying reproductive biology such as gene editing and single-cell technologies.