Advances in the application of induced pluripotent stem cells in pediatric diseases.

The optimal diagnosis and treatment of pediatric diseases depend on more adequate understanding of pathophysiology. The advent of induced pluripotent stem cells (iPSCs) has provided new strategies for the research and therapy of pediatric diseases. iPSCs are pluripotent stem cells induced by reprogramming of mature cells. Now they can be induced from many types of somatic cells (such as fibroblasts, peripheral blood mononuclear cells and urine cells).With the improvement of various reprogramming methods, its generation procedure is more and more optimized, and the use of small molecules to induce iPSCs is one of the research focus now. Due to their ability to differentiate into a variety of cells, combined with the development of gene editing technology, iPSCs have been increasingly favored in the modeling of diseases and cell therapy, especially hereditary diseases, and have achieved some success in clinical treatment. But before they can be widely used in clinical treatment, there are still some problems to be solved, such as tumorigenicity, immunogenicity and heterogeneity. This article reviewed the source of iPSCs, reprogramming technology, applications of iPSCs in common childhood diseases, current problems and prospects, in order to deepen the understanding of iPSCs and provide reference for in-depth research in field of exploring mechanisms of diseases and therapy of diseases.

Role of surfactant protein C in neonatal genetic disorders of the surfactant system: A case report.

RATIONALE: Respiratory distress syndrome (RDS) refers to the symptoms of progressive dyspnea and respiratory failure in newborns shortly after birth. The clinical and genetic characteristics of patients with neonatal RDS have not been extensively reported. PATIENT CONCERNS: A infant was in critical condition with repeated paroxysmal blood oxygen decline. Oxygen inhalation and noninvasive ventilator-assisted breathing relief were not effective. The etiology was unclear, and there was no family history of lung disease. Surface-active substance replacement therapy and positive pressure-assisted ventilation support were ineffective. DIAGNOSIS: The infant was clinically diagnosed with RDS. Genetic tests revealed a heterozygous missense mutation in the c.168 surfactant protein C (SFTPC) gene. INTERVENTIONS: Tracheal intubation was performed with invasive ventilator-assisted breathing, pulmonary surfactant was administered. Supportive treatment for liver protection and administration of a cardiotonic diuretic, vasodilator, human immunoglobulin (intravenous infusion), fresh frozen plasma, and suspended red blood cells were performed. OUTCOMES: The infant showed poor responses to respiratory and circulatory support, antibiotic treatment, and other treatment methods. The patient was discharged from hospital against the advice of us, cut off from us. The long-term prognosis of the patient after discharge remains unknown. LESSONS: SFTPC gene mutations may be an important risk factor for the development of common lung diseases. Because of the important roles of surfactant functions and metabolism, mutations in these genes can affect the production and function of pulmonary surfactant, leading to severe lung disease in term newborns.

A novel silent deletion, an insertion mutation and a nonsense mutation in the TCOF1 gene found in two Chinese cases of Treacher Collins syndrome.

Treacher Collins syndrome (TCS) is the most common and well-known craniofacial disorder caused by mutations in the genes involved in pre-rRNA transcription, which include the TCOF1 gene. This study explored the role of TCOF1 mutations in Chinese patients with TCS. Mutational analysis of the TCOF1 gene was performed in three patients using polymerase chain reaction and direct sequencing. Among these three patients, two additional TCOF1 variations, a novel 18 bp deletion and a novel 1 bp insertion mutation, were found in patient 1, together with a novel nonsense mutation (p.Ser476X) and a previously reported 4 bp deletion (c.1872_1875delTGAG) in other patients. Pedigree analysis allowed for prediction of the character of the mutation, which was either pathological or not. The 18 bp deletion of six amino acids, Ser-Asp-Ser-Glu-Glu-Glu (798*803), which was located in the CKII phosphorylation site of treacle, seemed relatively benign for TCS. By contrast, another novel mutation of c.1072_1073insC (p.Gln358ProfsX23) was a frameshift mutation and expected to result in a premature stop codon. This study provides insights into the functional domain of treacle and illustrates the importance of clinical and family TCS screening for the interpretation of novel sequence alterations.

Value of amplitude-integrated electroencephalograph in early diagnosis and prognosis prediction of neonatal hypoxic-ischemic encephalopathy.

UNLABELLED: To investigate value of amplitude-integrated electroencephalograph (aEEG) in early diagnosis and prediction of long-term prognosis of neonatal hypoxic-ischemic encephalopathy (HIE), 120 HIE Children were randomly assigned into aEEG group and control group (n = 60 per group). Children in each group were sub-divided into mild, moderate and severe HIE groups (n = 20 per group). 1, 3, 14 and 28 days after birth, aEEG was performed in aEEG group; 3, 14 and 28 days after birth, neonatal behavioral neurological assessment (NBNA) was done in both groups. Children who discharged were followed up at adjusted gestational age of 12 months with Denver Developmental Screening Test (DDST) and prognosis evaluation. RESULTS: aEEG manifestation was positively related to clinical severityb of HIE (r = 0.843, P < 0.01). On day 3 and 14, NBNA score was comparable between two groups (P > 0.05), but significant difference in NBNA score was noted on day 28 (P < 0.05). On day 3, 14 and 28, aEEG manifestation was positively associated with prognosis at adjusted gestational age of 12 months (r = 0.832, 0.857, 0.778, 0.743, P < 0.01). In aEEG group, disability rate was 13.8%, which was significantly lower than that in control group (23.2%); cure rate in aEEG group (60%) was significantly higher than that in control group (40%). Moreover, long-term prognosis was also dramatically different between aEEG group and control group (χ(2) = 4.107, P < 0.05). CONCLUSION: aEEG manifestation is significantly associated with clinical severity of HIE and may be helpful for early diagnosis of HIE. aEEG may be used to predict long term prognosis of HIE children.

[Establishment of regional active neonatal transport network].

OBJECTIVE: To evaluate the clinical function and significance of establishing a regional active neonatal transport network (ANTN) in Beijing. METHOD: The authors retrospectively studied intensive care and the role of ANTN system in management of critically ill neonates and compared the outcome of newborn infants transported to our NICU before and after we established standardized NICU and ANTN system (phase 1: July 2004 to June 2006 vs phase 2: July 2006 to May 2008). RESULT: The number of neonatal transport significantly increased from 587 during phase 1 to 2797 during phase 2. Success rate of transport and the total cure rate in phase 2 were 97.85% and 91.99% respectively, which were significantly higher than those in phase 1 (94.36% and 88.69%, respectively, P < 0.01). The neonatal mortality significantly decreased in phase 2 compared with that in phase 1 (2.29% vs 4.31%, P < 0.01). The capacity of our NICU was enlarged following the development of ANTN. There are 200 beds for level 3 infants in phase 2, but there were only 20 beds in phase 1. Significantly less patients in the phase 2 had hypothermia, acidosis and the blood glucose instability than those in phase 1 (P < 0.01, 0.05, 0.01 and 0.05, respectively). The proportion of preterm infants transported to our NICU were higher in phase 2 compared with that in phase 1, especially infants whose gestational age was below 32 weeks. The proportions of asphyxia and respiratory distress syndrome were lower in phase 2 than that in phase 1, but the total cure rates of these two diseases had no significant changes between the two phases. The most important finding was that the improvement of outcome of premature infants and those with asphyxia and aspiration syndrome was noted following the development of ANTN. CONCLUSION: Establishing regional ANTN for a tertiary hospital is very important to elevate the total level in management of critically ill newborn infants. It plays a very important role in reducing mortality and improving total outcomes of newborn infants. There are still some problems remained to solve after four years practice in order to optimize the ANTN to meet needs of the development of neonatology.

[Preliminary study on maturity of retinal vascularization in premature infants].

OBJECTIVE: To study the extent of retinal vascular development and influencing factors at birth and the relation between retinal vascularization and retinopathy of prematurity (ROP). METHODS: From October, 2006 to December 2006, retinal vascularization was screened and evaluated in 84 neonates at different weeks of gestation and birth weights (BWs), had dilated fundus evaluation for zone of retinal vascularization by the 130 degrees lens of a digital fundus camera. The infants' pupils were dilated with 2.5% phenylephrine and 0.5% cyclopentolate eye drops. The study cohort was divided into subgroups depending on the weeks of gestation and birth weights. The control group consisted of healthy term infants. Maternal and neonatal factors were ascertained and analysed. RESULTS: Vascularization up to zone I and II was considered to be immature retina; vascularization up to zone III or beyond was considered to be mature retina. In this study, 11 of 12 infants who were born at < 30 weeks of gestation, 12 of 26 infants who were born at < 31 approximately 33 weeks of gestation, 1 of 26 babies who were born at < 34 approximately 36 weeks of gestation and none of 20 babies who were born at < 37-40 weeks of gestation had immature retina; 12 of 15 babies at < 1500 g BW, 8 of 14 infants at 1500 g < BW < 1700 g, 4 of 11 infants at 1700 g < BW < 2000 g and of 44 infants at > 2000 g BW had immature retina. Those infants who were born at > 34 weeks of gestational age and at > 2000 g BW had mature retina. Infants who were born between 31 to 34 weeks of gestation and at 1501 to 2000 g BW had variable extent of retinal vascularization at birth. Vascularization was associated with postconceptional age (F = 31.9193, P = 0.000), birth weight (F = 32.4532, P = 0.000), anemia (F = 36.9391, P = 0.000), surfactant (F = 24.000, P = 0.0000), poor nutrition (F = 4.184, P = 0.041), RDS (F = 17.6191, P = 0.000), cesarean delivery (F = 10.972, P = 0.0022) and oxygen > 48 h (F = 22.076, P = 0.0000). Vascularization was affected mainly by the postconceptional age (95% CI = 1.57-261.728, P = 0.021). At last, 15/24 infants with immature retina developed ROP while none of the infants with mature retina developed ROP (chi2 = 45.1087, P = 0.000). CONCLUSION: There is considerable variability in the extent of retinal vascularization in infants who we born between 31 to 34 weeks of gestation. Modifiable maternal and fetal factors could influence extent of vascularization at birth. Immature retina is the critical factor of ROP. Gestational age is the main factor of the immature retina in premature infants.

[Effect of basic fibroblast growth factor on expression of protein and mRNA of bone morphogenetic protein 4 in hypoxic-ischemic brain damage in newborn rats].

OBJECTIVE: To investigate the effect of basic fibroblast growth factor(bFGF) on expression of protein and mRNA of bone morphogenetic protein 4 in hypoxic-ischemic brain damage (HIBD) in newborn rats. METHOD: One hundred and twenty 7 days old neonatal rats were randomly divided into control group, hypoxic-ischemic brain damage and interventional group of bFGF, each having forty neonatal rats. After HIBD model was established, bFGF was given to interventional group by peritoneal injection for 5 continuous days. Every group was randomly divided into 7 days, 14 days, 21 days and 28 days group, according to the time of sacrifice. BMP4 protein in hippocampus was determined with immunohistochemical method. Messenger RNA of BMP4 were determined with in situ hybridization. Apoptosis of nerve cell was determined with TUNEL. Intergroup or intragroup comparisons were performed with analysis of variance. RESULT: On the days 7 and 14, expression of BMP4 protein in hippocampus was higher in interventional group of bFGF than in HIBD while expression of BMP4 protein in interventional group of bFGF and HIBD was lower on day 7 than on day 14. Expression of BMP4 protein on the days 21 and 28 had no significant difference among three groups. mRNA expression of BMP4 in interventional group of bFGF and HIBD was significantly higher in hippocampus than in control group. On the day 14, BMP4 mRNA in hippocampus widely expressed in HIBD while BMP4 mRNA only expressed in CA1 in interventional group of bFGF. Expression of BMP4 mRNA in hippocampus on the affected side decreased from the time of killing on 28th day while there was no significant change in interventional group of bFGF. Apoptosis of neural cells at the time of sacrifice on day 7 was lower in interventional group of bFGF than that in HIBD group (F=9.010, P<0.01). Apoptotic neural cells was higher in bFGF and HIBD groups at the time of killing on days 14, 21 and 28 than that on day 7 but that the bFGF group had less apoptotic neural cells than HIBD group (F=9.202, 7.932, 14.985, P<0.01). CONCLUSIONS: bFGF has a neurorestoration effect, which promotes expression of BMP4 protein and BMP4 mRNA in hippocampus of HIBD and inhibit apoptosis of neural cells.

[Relationship between reduced expression of surfactant protein B and neonatal respiratory distress syndrome in twenty Han ethnic group neonates in China].

OBJECTIVE: To investigate possible relationship between expression of surfactant protein B (SP-B) gene product and neonatal respiratory distress syndrome (NRDS) in Han ethnic group. METHOD: Unrelated 20 cases with NRDS of Han ethnic group were selected as NRDS group while unrelated 20 diseases cases of Han ethnic group with diseases were selected as control group. The cases in the control group had congenital heart disease or bronchopulmonary dysplasia or persistent pulmonary hypertension. Blood sample was taken from every case. Lung tissues were taken from the patients who died half an hour after death in the two groups. Expression of SP-B in lung tissue was determined with immunohistochemical tecnique. Genetic deficiency variant of SP-B intron IV was screened with polymerase chain reaction (PCR). RESULTS: Two cases at gestational age 26 weeks and one case at gestational age 34 weeks and two cases at gestational age 42 weeks of NRDS groups had lower level expression of SP-B in lung tissue than those at the same age of NRDS. Expression of SP-B in lung tissue of control group increased with gestational age, but no such phenomenon was found in NRDS group. Further, two cases at gestational age 42 weeks of NRDS group had genetic deficiency variant of SP-B intron IV with gene analysis of five cases who had lower expression of SP-B. Clinical data suggest that patients at 42 weeks of gestational age had severe illness. CONCLUSIONS: Decrease of SP-B expression may participate in occurrence of NRDS, genetic deficiency variant of SP-B intron IV exists in the NRDS cases of Han ethnic group of China.

The role of antenatal corticosteroids for improving the maturation of choroid plexus capillaries in fetal mice.

We investigated whether the use of antenatal corticosteroids could improve the maturation of choroid plexus capillaries in fetal mice. The study was carried out in two groups of preterm mice. The study group consisted of pregnant mice that received dexamethasone at a dose of 4 mg/kg intraperitoneally. This group was further subdivided into four subgroups according to the timing of steroid administration as follows: day 13, day 14, day 15, and day 16 of pregnancy, and each subgroup included 12 premature mice. All animals received a second injection 24 hours after the first injection. The control group was given normal saline. The pregnant mice were operated on to obtain premature mice. The choroid plexus capillaries were assessed for integrity and thickness of their basement membranes by electronmicroscopy. We found that, in the study group, the maturity of the basement membrane of the choroid plexus capillaries was more precise, in that capillaries were better in control animals at term for the following reasons: (a) the basement membrane becomes more intact, (b) thickness of the basement membrane increased, and (c) the protein particles become tighter. Antenatal dexamethasone improved the maturation of the choroid plexus capillaries in fetal mice but also decreased the incidence of periventricular-intraventricular hemorrhage (PIVH). Using two doses of steroids had no influence on birthweight or brainweight of the mice. In conclusion, these findings provide an experimental basis for the use of antenatal steroids for decreasing the incidence of PIVH in premature infants.

[A multivariate analysis of prevalence and risk factors of retinopathy of prematurity].

OBJECTIVE: To investigate the prevalence and risk factors of retinopathy of prematurity (ROP). METHODS: This investigation involved 125 premature infants admitted in the neonate intensive unit between July 1st, 2006 and Feb 1st, 2007, who were less than 37 weeks of postconceptional age, or more than 37 weeks but with birth weight <2500 g. At the fourth postnatal week or the corrected gestational age of 32 to 34 weeks, the infants underwent ROP examination of both eyes using RetCam digital retinal camera. Diagnosis and staging of ROP were established according to the international guidelines, with another 20 full-term infants as the control group. RESULTS: All the 125 infants completed the follow up. The prevalence of ROP in the premature group was 6.4%, while no ROP was found in the control group. Of the premature infants, the prevalence of ROP in infants with birth weight

[Experimental study on growth, proliferation and differentiation of neural stem cell from subventricular zone of human fetal brain at different gestational age].

OBJECTIVE: To study growth characteristics of neural stem cells (NSCs) from subventricular zone (SVZ) of the different human fetal brain at different gestational age and to provide experimental and theoretical evidences for clinical application of NSCs for treatment of certain diseases. METHODS: Ninety human embryos at gestational age 16 - 36 weeks were collected and were divided into six groups according to gestational age: 16 w, 20 w, 24 w, 28 w, 32 w and 36 w. Each group had 15 embryos and brain tissues were taken from each embryo's SVZ. All subjects had congenital heart disease or digestive tract abnormity diagnosed with B ultrasound at antepartum, but none had abnormal development of brain. Pregnant mother and her husband desire termination of pregnancy. The morphology, existing mode and the number of neural stem cells in subventricular zone were examined with immunohistochemical method. The NSCs in subventricular zone were cultured, passaged and differentiated with cell culture technique, then were identified with immunohistochemical method. RESULTS: NSCs in SVZ from the different human fetal brain existed in a scattered manner in the network formed by stellate cells, NSCs had round, ellipse and fusiform shape, especially in stellate shape. NSCs had larger and smaller size and distributed in dense or scattered forms, each having zero to two enations, most had one or two. NSCs had less cytoplasm. The nucli of the NSCs had a round shape with loose chromatin and 1 - 4 nucleoli. Most of NSCs existed in singular scattered form, some of them showed symmetrical or asymmetrical division, some of them showed synaptic connection with other NSCs. The number of NSCs in SVZ from groups with different fetal age decreased with increasing gestational age (chi(2) = 4644.602, P < 0.01). NSCs in SVZ from the different human fetal brain cultured with serum-free medium formed typical neurospheres in suspension. The cells could be passaged continuously, and could express nestin antigen. Serum-contained medium induced neural stem cells to differentiate and express specific antigens of neuron, astrocyte and oligodendrocyte. CONCLUSIONS: NSCs existed in SVZ of human embryos at different gestational age. There are differences in morphology, existing pattern and the number of NSCs in SVZ at different gestational age. NSCs in SVZ at different gestational age may be cultured in vitro.

[Changes of neural stem cells in neonatal rat model of hypoxic-ischemic encephalopathy].

OBJECTIVE: To establish a neonatal rat model of hypoxic-ischemic encephalopathy and clarify the changing features of neural stem cells (NSCs) in episodes of hypoxic-ischemic encephalopathy (HIE) so as to provide experimental and theoretical evidences for treating HIE by applying NSCs at the appropraite time. METHODS: Totally 210 neonatal rats aged 7 d were divided randomly into three groups, normal control group, hypoxic group and hypoxic-ischemic group with 70 rats in each. According to the time of sacrefice, 70 rats of every group were further divided randomly into seven groups including third hour (3 h), the sixth hour (6 h), first day (1 d), third day (3 d), the seventh day (7 d), the fourteenth day (14 d) and the twenty-first day (21 d), with 10 rats in each subgroup. The left common carotid artery of the neonatal rats in hypoxic-ischemic group was ligated and those in the hypoxic group were subjected to inhalation of 8% oxygen for 2.5 h. NSCs from brain tissues of the rats of the three groups were determined with HE staining and immunohistochemical method under light microscope. RESULTS: Most of neonatal rats in hypoxic-ischemic group behaved turning to the left stably 1 h after normal concentration of oxygen was given. In hypoxic-ischemic group, slight brain injury occurred at 3 h, severe brain injury appeared at 1 d, glial cells proliferated at 3 d and 7 d, brain atrophy was found at 14 d and 21 d. NSCs existed in brain tissues of rats in all the three groups. NSCs in normal control and the hypoxic group mainly distributed in hippocampus, subventricular tissues, striatum and cortex. But NSCs in hippocampus located in layers of molecule, cone cell and inner granular cell. NSCs in hypoxic-ischemic group showed obvious regional distribution, less in the regions with pathological changes. At 3 h, 6 h and 14 d, there was no difference in the number of NSCs between hypoxi and hypoxic-ischemic group. At 1 d, 3 d and 7 d, there was a highly significant difference in the number of NSCs between hypoxic and hypoxic-ischemic group. At 21 d, there was a significant difference in the number of NSCs between hypoxic and hypoxic-ischemic group, meanwhile, there was a significant difference in the number of NSCs between control and hypoxic group. At 3 d, there was a very significant difference in the number of NSCs between control and hypoxic-ischemic group. At 7 d and 21 d points, there was a highly significant difference in the number of NSCs between control and hypoxic group. CONCLUSION: The neonatal rat model of HIE was successfully established. NSCs increased in earlier period and decreased in later period of HIE, ultimately, NSCs located in the injured regions died one after anotner. Hypoxia induces NSCs' proliferation.

[Effects of neonatal calf serum on differentiation of human fetal neural stem cells in the hippocampus].

OBJECTIVE: To investigate the effects of neonatal calf serum on the differentiation of human neural stem cells in the hippocampus. METHODS: The effects of various concentrations of neonatal calf serum on the differentiation of human fetal neural stem cells in the hippocampus were observed in cell culture experiment and immunocytochemical staining. RESULTS: Neonatal calf serum efficiently induced the differentiation of human fetal neural stem cells into neurons and astrocytes, whose amount varied between serum-free cell culture and the culture with neonatal calf serum of different concentrations. As the concentration of neonatal calf serum increased, the differentiation of human fetal neural stem cells shifted toward astrocytes, while the differentiation into neurons was decreased. CONCLUSION: Neonatal calf serum causes alteration of the ratio between neurons and glial cells differentiated from human neural stem cells in the hippocampus.

[Phenotype analysis of alpha1-antichymotrypsin in the Guangzhou Han population].

OBJECTIVE: To understand the phenotype distribution profile of alpha1-antichymotrypsin (alpha1-ACT) in the Han population in Guangzhou municipality so as to decide whether geographical factors affect the distribution of alpha1-ACT phenotypes. METHODS: Polyacrylamide gel isoelectric focusing followed by immunofixation techniques was employed for examining the alpha1-ACT phenotypes in 200 unrelated individuals randomly selected from the Guangzhou Han residents. RESULTS: Three phenotypes of alpha1-ACT were identified in this subject cohort, which were identical to those identified in Chongqing but with different distributions of the phenotype frequencies. CONCLUSION: Geographical factors may affect the distribution of the phenotype frequencies of alpha1-ACT, which is a promising genetic marker for the pursuit of human genetics.