Vitamin C- and Valproic Acid-Induced Fetal RPE Stem-like Cells Recover Retinal Degeneration via Regulating SOX2.

Retinal pigment epithelial (RPE) cell replacement therapy has provided promising outcomes in the treatment of retinal degenerative diseases (RDDs), but the resulting limited visual improvement has raised questions about graft survival and differentiation. Through combined treatment with vitamin C and valproic acid (together, VV), we activated human fetal RPE (fRPE) cells to become highly proliferative fetal RPE stem-like cells (fRPESCs). In this study, we report that SOX2 (SRY-box 2) activation contributed to mesenchymal-epithelial transition and elevated the retinal progenitor and mesenchymal stromal markers expressions of fRPESCs. These fRPESCs could differentiate into RPE cells, rod photoreceptors, and mesenchymal lineage progenies under defined conditions. Finally, fRPESCs were transplanted into the subretinal space of an RDD mouse model, and a photoreceptor rescue benefit was demonstrated. The RPE and rod photoreceptor differentiation of transplanted fRPESCs may account for the neural retinal recovery. This study establishes fRPESCs as a highly proliferative, multi-lineage differentiation potential (including RPE, rod photoreceptor, and mesenchymal lineage differentiation), mesenchymal-to-epithelial-transitioned retinal stem-like cell source for cell-based therapy of RDDs.

In Utero Transplantation of Expanded Autologous Amniotic Fluid Stem Cells Results in Long-Term Hematopoietic Engraftment.

In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been proposed as a strategy for the prenatal treatment of congenital hematological diseases. However, levels of long-term hematopoietic engraftment achieved in experimental IUT to date are subtherapeutic, likely due to host fetal HSCs outcompeting their bone marrow (BM)-derived donor equivalents for space in the hematopoietic compartment. In the present study, we demonstrate that amniotic fluid stem cells (AFSCs; c-Kit+/Lin-) have hematopoietic characteristics and, thanks to their fetal origin, favorable proliferation kinetics in vitro and in vivo, which are maintained when the cells are expanded. IUT of autologous/congenic freshly isolated or cultured AFSCs resulted in stable multilineage hematopoietic engraftment, far higher to that achieved with BM-HSCs. Intravascular IUT of allogenic AFSCs was not successful as recently reported after intraperitoneal IUT. Herein, we demonstrated that this likely due to a failure of timely homing of donor cells to the host fetal thymus resulted in lack of tolerance induction and rejection. This study reveals that intravascular IUT leads to a remarkable hematopoietic engraftment of AFSCs in the setting of autologous/congenic IUT, and confirms the requirement for induction of central tolerance for allogenic IUT to be successful. Autologous, gene-engineered, and in vitro expanded AFSCs could be used as a stem cell/gene therapy platform for the in utero treatment of inherited disorders of hematopoiesis. Stem Cells 2019;37:1176-1188.

Stem Cell Therapy as a Treatment for Osteogenesis Imperfecta.

PURPOSE OF REVIEW: Osteogenesis imperfecta (OI) is a chronic disease with few treatment options available. The purpose of this review is to provide an overview on treating OI with mesenchymal stem cells (MSC). RECENT FINDINGS: Off-the-shelf MSC have a good safety profile and exhibit multilineage differentiation potential and a low immunogenic profile and are easy to manufacture. Their ability to migrate, engraft, and differentiate into bone cells, and also to act via paracrine effects on the recipient's tissues, makes MSC candidates as a clinical therapy for OI. Due to their high osteogenic potency, fetal MSC offer an even higher therapeutic potential in OI compared with MSC derived from adult sources. Preclinical and initial clinical data support the use of MSC in treating OI. The characteristics of MSC make them of great interest in treating OI. MSC may be safely transplanted via intravenous administration and show potential positive clinical effects.

Efficient decellularization of equine tendon with preserved biomechanical properties and cytocompatibility for human tendon surgery indications.

Chronic and acute tendon injuries are frequent afflictions, for which treatment is often long and unsatisfactory. When facing extended injuries, matrices and scaffolds with sufficient biomechanical properties are required for surgical repair and could additionally serve as supports for cellular therapies to improve healing. In this study, protocols of either commonly used detergents only (SDS 1%, Triton 1%, TBP 1%, and Tween-20 1%) or a combination of freeze/thaw (F/T) cycles with decellularization agents (NaCl 1M, ddH2 O) were evaluated for the decellularization of horse equine superficial digital flexor tendon (SDFT) for hand flexor or extensor tendon reconstruction. Decellularization efficiency was assessed microscopically by histological staining (HE, DAPI) and DNA quantification. Macroscopical structure and biomechanical integrity of the tendon matrices were further assessed by gross observation, histological staining (SR), and mechanical testing (ultimate strain and stress, Young's modulus, energy to failure) for select protocols. Decellularization with hypertonic NaCl 1M in association with F/T cycles produced the most robust tendon matrices, which were nontoxic after 10 days for subsequent recellularization with human fetal progenitor tendon cells (hFPTs). This standardized protocol uses a less aggressive decellularization agent than current practice, which allows subsequent reseeding with allogenic cells, therefore making them very suitable and bioengineered tendon matrices for human tendon reconstruction in the clinic.

Effect of Transplantation of Neural Stem and Progenitor Cells on Memory in Animals with Alzheimer's Type Neurodegeneration.

The effects of systemic and intracerebral transplantation of human fetal neural stem and progenitor cells were studied on the model of olfactory bulbectomy in mice with developing signs of sporadic Alzheimer's disease. It was found that transplantation of these cells at certain stages of disease development contributed to improvement of spatial memory and preservation of hippocampal neurons in these animals.

Therapeutic abortion and ectopic pregnancy: alternative sources for fetal stem cell research and therapy in Iran as an Islamic country.

Regenerative medicine as a background of stem cell research and therapy has a long history. A wide variety of diseases including Parkinson's disease, heart diseases, multiple sclerosis, spinal cord injury, diabetes mellitus and etc. are candidate to be treated using different types of stem cells. There are several sources of stem cells such as bone marrow, umbilical cord, peripheral blood, germ cells and the embryo/fetus tissues. Fetal stem cells (FSCs) and embryonic stem cells (ESCs) have been described as the most potent stem cell source. Although their pluri- or multipotent properties leads to promising reports for their clinical applications, owning to some ethical and legal obstacles in different communities such as Muslim countries, care should be taken for therapeutic applications of FSCs and ESCs. Derivation of these cell types needs termination of pregnancy and embryo or fetus life that is prohibited according to almost all rules and teaches in Muslim communities. Abortion and termination of pregnancy under a normal condition for the procurement of stem cell materials is forbidden by nearly all the major world religions such as Islam. Legislated laws in the most of Muslim countries permit termination of pregnancy and abortion only when the life of the mother is severely threatened or when continuing pregnancy may lead to the birth of a mentally retarded, genetically or anatomically malformed child. Based on the rules and conditions in Islamic countries, finding an alternative and biologically normal source for embryonic or fetal stem cell isolation will be too difficult. On the one hand, Muslim scientists have the feasibility for finding of genetically and anatomically normal embryonic or fetal stem cell sources for research or therapy, but on the other hand they should adhere to the law and related regional and local rules in all parts of their investigation. The authors suggest that the utilization of ectopic pregnancy (EP) conceptus, extra-embryonic tissues, and therapeutic abortion materials as a valuable source of stem cells for research and medical purposes can overcome limitations associated with finding the appropriate stem cell source. Pregnancy termination because of the mentioned subjects is accepted by almost all Islamic laws because of maternal lifesaving. Also, there are no ethical or legal obstacles in the use of extra-embryonic or EP derived tissues which lead to candidate FSCs as a valuable source for stem cell researches and therapeutic applications.

Impact of Cryopreservation on Caprine Fetal Adnexa Derived Stem Cells and Its Evaluation for Growth Kinetics, Phenotypic Characterization, and Wound Healing Potential in Xenogenic Rat Model.

This study was conducted to know the impact of cryopreservation on caprine fetal adnexa derived mesenchymal stem cells (MSCs) on the basic stem cell characteristics. Gravid caprine uteri (2-3 months) were collected from local abattoir to derive (amniotic fluid [cAF], amniotic sac [cAS], Wharton's jelly [cWJ], and cord blood [cCB]) MSCs and expanded in vitro. Cells were cryopreserved at 3rd passage (P3) using 10% DMSO. Post-thaw viability and cellular properties were assessed. Cells were expanded to determine growth kinetics, tri-lineage differentiation, localization, and molecular expression of MSCs and pluripotency markers; thereafter, these cells were transplanted in the full-thickness (2 × 2cm2 ) rat skin wound to determine their wound healing potential. The post-thaw (pt) growth kinetics study suggested that cWJ MSCs expanded more rapidly with faster population doubling time (PDT) than that of other fetal adnexa MSCs. The relative mRNA expression of surface antigens (CD73, CD90, and CD 105) and pluripotency markers (Oct4, KLF, and cMyc) was higher in cWJ MSCs in comparison to cAS, cAF, and cCB MSCs post-thaw. The percent wound contraction on 7th day was more than 50% for all the MSC-treated groups (pre and post-thaw), against 39.55% in the control group. On day 28th, 99% and more wound contraction was observed in cAF, cAF-pt, cAS-pt, cWJ, cWJ-pt, and cCB, MSCs with better scores for epithelization, neovascularization, and collagen characteristics at a non-significant level. It is concluded that these MSCs could be successfully cryopreserved without altering their stemness and wound healing properties. J. Cell. Physiol. 232: 2186-2200, 2017. © 2016 Wiley Periodicals, Inc.

Anti-muscarinic adjunct therapy accelerates functional human oligodendrocyte repair.

Therapeutic repair of myelin disorders may be limited by the relatively slow rate of human oligodendrocyte differentiation. To identify appropriate pharmacological targets with which to accelerate differentiation of human oligodendrocyte progenitors (hOPCs) directly, we used CD140a/O4-based FACS of human forebrain and microarray to hOPC-specific receptors. Among these, we identified CHRM3, a M3R muscarinic acetylcholine receptor, as being restricted to oligodendrocyte-biased CD140a(+)O4(+) cells. Muscarinic agonist treatment of hOPCs resulted in a specific and dose-dependent blockade of oligodendrocyte commitment. Conversely, when hOPCs were cocultured with human neurons, M3R antagonist treatment stimulated oligodendrocytic differentiation. Systemic treatment with solifenacin, an FDA-approved muscarinic receptor antagonist, increased oligodendrocyte differentiation of transplanted hOPCs in hypomyelinated shiverer/rag2 brain. Importantly, solifenacin treatment of engrafted animals reduced auditory brainstem response interpeak latency, indicative of increased conduction velocity and thereby enhanced functional repair. Therefore, solifenacin and other selective muscarinic antagonists represent new adjunct approaches to accelerate repair by engrafted human progenitors.

Brain repair and reprogramming: the route to clinical translation.

The adult brain has a very limited capacity for generation of new neurons, and neurogenesis only takes place in restricted regions. Some evidence for neurogenesis after injury has been reported, but few, if any, neurons are replaced after brain injury or degeneration, and the permanent loss of neurons leads to long-term disability and loss of brain function. For decades, researchers have been developing cell transplantation using exogenous cell sources for brain repair, and this method has now been shown to successfully restore lost function in experimental and clinical trials. Here, we review the development of cell-replacement strategies for brain repair in Parkinson's disease using the example of human foetal brain cells being successfully translated from preclinical findings to clinical trials. These trials demonstrate that cell-replacement therapy is a viable option for patients with Parkinson's disease, but more importantly also show how the limited availability of foetal cells calls for development of novel cell sources and methods for generating new neurons for brain repair. We focus on new stem cell sources that are on the threshold of clinical application for brain repair and discuss emerging cellular reprogramming technologies. Reviewing the current status of direct neural conversion, both in vitro and in vivo, where somatic cells are directly reprogrammed into functional neurons without passing through a stem cell intermediate, we conclude that both methods result in the successful replacement of new neurons that mature and integrate into the host brain. Thus, this new field shows great promise for future brain repair, although much work is still needed in preclinical animal models before it can be seriously considered for clinical applications.

A competitive advantage by neonatally engrafted human glial progenitors yields mice whose brains are chimeric for human glia.

Neonatally transplanted human glial progenitor cells (hGPCs) densely engraft and myelinate the hypomyelinated shiverer mouse. We found that, in hGPC-xenografted mice, the human donor cells continue to expand throughout the forebrain, systematically replacing the host murine glia. The differentiation of the donor cells is influenced by the host environment, such that more donor cells differentiated as oligodendrocytes in the hypomyelinated shiverer brain than in myelin wild-types, in which hGPCs were more likely to remain as progenitors. Yet in each recipient, both the number and relative proportion of mouse GPCs fell as a function of time, concomitant with the mitotic expansion and spread of donor hGPCs. By a year after neonatal xenograft, the forebrain GPC populations of implanted mice were largely, and often entirely, of human origin. Thus, neonatally implanted hGPCs outcompeted and ultimately replaced the host population of mouse GPCs, ultimately generating mice with a humanized glial progenitor population. These human glial chimeric mice should permit us to define the specific contributions of glia to a broad variety of neurological disorders, using human cells in vivo.

The use of human amniotic fluid stem cells as an adjunct to promote pulmonary development in a rabbit model for congenital diaphragmatic hernia.

OBJECTIVE: This study aimed to evaluate the potential benefit of intra-tracheal injection of human amniotic fluid stem cells (hAFSC) on pulmonary development combined with TO in a rabbit model for CDH. METHODS: In time-mated pregnant does a left diaphragmatic defect was created at d23 (term = 31). At d28, previously operated fetuses were assigned to either TO and injection with 70 µL of phosphate buffered saline (PBS) or 1.0 × 10(6) c-Kit positive hAFSC expressing LacZ or were left untouched (CDH). Harvesting was done at d31 to obtain their lung-to-body weight ratio (LBWR), airway and vascular lung morphometry, X-gal staining and immunohistochemistry for Ki67 and surfactant protein-B (SP-B). RESULTS: CDH-induced pulmonary hypoplasia is countered by TO + PBS, this reverses LBWR, mean terminal bronchiole density (MTBD) and medial thickness to normal. The additional injection of hAFSC decreases MTBD and results in a non-significant decrease in muscularization of intra-acinary vessels. There were no inflammatory changes and LacZ positive hAFSC were dispersed throughout the lung parenchyma 4 days after injection. CONCLUSION: HAFSC exert an additional effect on TO leading to a decrease in MTBD, a measure of alveolar number surrounding the terminal bronchioles, without signs of toxicity. © 2015 John Wiley & Sons, Ltd.

Clinical Trial of Human Fetal Brain-Derived Neural Stem/Progenitor Cell Transplantation in Patients with Traumatic Cervical Spinal Cord Injury.

In a phase I/IIa open-label and nonrandomized controlled clinical trial, we sought to assess the safety and neurological effects of human neural stem/progenitor cells (hNSPCs) transplanted into the injured cord after traumatic cervical spinal cord injury (SCI). Of 19 treated subjects, 17 were sensorimotor complete and 2 were motor complete and sensory incomplete. hNSPCs derived from the fetal telencephalon were grown as neurospheres and transplanted into the cord. In the control group, who did not receive cell implantation but were otherwise closely matched with the transplantation group, 15 patients with traumatic cervical SCI were included. At 1 year after cell transplantation, there was no evidence of cord damage, syrinx or tumor formation, neurological deterioration, and exacerbating neuropathic pain or spasticity. The American Spinal Injury Association Impairment Scale (AIS) grade improved in 5 of 19 transplanted patients, 2 (A → C), 1 (A → B), and 2 (B → D), whereas only one patient in the control group showed improvement (A → B). Improvements included increased motor scores, recovery of motor levels, and responses to electrophysiological studies in the transplantation group. Therefore, the transplantation of hNSPCs into cervical SCI is safe and well-tolerated and is of modest neurological benefit up to 1 year after transplants. This trial is registered with Clinical Research Information Service (CRIS), Registration Number: KCT0000879.

Effect of osteopontin in regulating bone marrow mesenchymal stem cell treatment of skin wounds in diabetic mice.

BACKGROUND: We aimed to investigate the role of osteopontin in regulating mesenchymal stem cells transplanted to promote wound healing in diabetic mice. METHODS: The mesenchymal stem cells of osteopontin knock-out (KO) and wild-type (WT) mice were isolated separately for in vitro culture and characterization. A skin wound on the back of mice was established by skin punching. In 27 osteopontin KO male mice, induced diabetes mellitus was via intraperitoneal injection of streptozotocin. 9 normal mice were used as controls. The mice were divided into four groups and injected with Dulbecco's modified Eagle's medium (DMEM) or mesenchymal stem cells via the tail vein: A (diabetic mice injected with DMEM), B (diabetic mice injected with osteopontin KO mesenchymal stem cells), C (diabetic mice injected with WT mesenchymal stem cells), D (normal mice injected with DMEM). The healing times and closure rates of skin wounds were recorded. The microvessel density of healing wounds was measured, and the localized expression of osteopontin was identified by western blotting and immunohistochemistry. The migration of mesenchymal stem cells was observed on normal mice with skin wound injected with mesenchymal stem cells of C57BL6~GFP transgenic mice, which show green fluorescent under UV light. RESULTS: Compared with normal mice, the healing time of wounds in the mice with diabetes and osteopontin KO was significantly prolonged (p < 0.01). After transplanting osteopontin KO mesenchymal stem cells, the healing time was slightly shorter. Meanwhile, the healing time was significantly shorter after transplanted with WT mesenchymal stem cells and more significant neovascularization at healing wounds (p < 0.05). The expression of osteopontin in local healing wounds after transplantation of WT mesenchymal stem cells was demonstrated with western blotting and immunohistochemistry. After 4 days, the green fluoresces were noted on the wounds of mice injected with mesenchymal stem cells of fluorescent mice. CONCLUSIONS: Mesenchymal stem cells can migrate to wound sites, and osteopontin plays a regulatory role in mesenchymal stem cells promoting the healing of diabetic skin wounds.

Phenotypic and in vivo functional characterization of immortalized human fetal liver cells.

We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.

An exploratory open-label multicentre phase I/II trial evaluating the safety and efficacy of postnatal or prenatal and postnatal administration of allogeneic expanded fetal mesenchymal stem cells for the treatment of severe osteogenesis imperfecta in infants and fetuses: the BOOSTB4 trial protocol.

INTRODUCTION: Severe osteogenesis imperfecta (OI) is a debilitating disease with no cure or sufficiently effective treatment. Mesenchymal stem cells (MSCs) have good safety profile, show promising effects and can form bone. The Boost Brittle Bones Before Birth (BOOSTB4) trial evaluates administration of allogeneic expanded human first trimester fetal liver MSCs (BOOST cells) for OI type 3 or severe type 4. METHODS AND ANALYSIS: BOOSTB4 is an exploratory, open-label, multiple dose, phase I/II clinical trial evaluating safety and efficacy of postnatal (n=15) or prenatal and postnatal (n=3, originally n=15) administration of BOOST cells for the treatment of severe OI compared with a combination of historical (1-5/subject) and untreated prospective controls (≤30). Infants<18 months of age (originally<12 months) and singleton pregnant women whose fetus has severe OI with confirmed glycine substitution in COL1A1 or COL1A2 can be included in the trial.Each subject receives four intravenous doses of 3×106/kg BOOST cells at 4 month intervals, with 48 (doses 1-2) or 24 (doses 3-4) hours in-patient follow-up, primary follow-up at 6 and 12 months after the last dose and long-term follow-up yearly until 10 years after the first dose. Prenatal subjects receive the first dose via ultrasound-guided injection into the umbilical vein within the fetal liver (16+0 to 35+6 weeks), and three doses postnatally.The primary outcome measures are safety and tolerability of repeated BOOST cell administration. The secondary outcome measures are number of fractures from baseline to primary and long-term follow-up, growth, change in bone mineral density, clinical OI status and biochemical bone turnover. ETHICS AND DISSEMINATION: The trial is approved by Competent Authorities in Sweden, the UK and the Netherlands (postnatal only). Results from the trial will be disseminated via CTIS, ClinicalTrials.gov and in scientific open-access scientific journals. TRIAL REGISTRATION NUMBERS: EudraCT 2015-003699-60, EUCT: 2023-504593-38-00, NCT03706482.

Safe genetic modification of cardiac stem cells using a site-specific integration technique.

BACKGROUND: Human cardiac progenitor cells (hCPCs) are a promising cell source for regenerative repair after myocardial infarction. Exploitation of their full therapeutic potential may require stable genetic modification of the cells ex vivo. Safe genetic engineering of stem cells, using facile methods for site-specific integration of transgenes into known genomic contexts, would significantly enhance the overall safety and efficacy of cellular therapy in a variety of clinical contexts. METHODS AND RESULTS: We used the phiC31 site-specific recombinase to achieve targeted integration of a triple fusion reporter gene into a known chromosomal context in hCPCs and human endothelial cells. Stable expression of the reporter gene from its unique chromosomal integration site resulted in no discernible genomic instability or adverse changes in cell phenotype. Namely, phiC31-modified hCPCs were unchanged in their differentiation propensity, cellular proliferative rate, and global gene expression profile when compared with unaltered control hCPCs. Expression of the triple fusion reporter gene enabled multimodal assessment of cell fate in vitro and in vivo using fluorescence microscopy, bioluminescence imaging, and positron emission tomography. Intramyocardial transplantation of genetically modified hCPCs resulted in significant improvement in myocardial function 2 weeks after cell delivery, as assessed by echocardiography (P=0.002) and MRI (P=0.001). We also demonstrated the feasibility and therapeutic efficacy of genetically modifying differentiated human endothelial cells, which enhanced hind limb perfusion (P<0.05 at day 7 and 14 after transplantation) on laser Doppler imaging. CONCLUSIONS: The phiC31 integrase genomic modification system is a safe, efficient tool to enable site-specific integration of reporter transgenes in progenitor and differentiated cell types.

Transplantation of human fetal blood stem cells in the osteogenesis imperfecta mouse leads to improvement in multiscale tissue properties.

Osteogenesis imperfecta (OI or brittle bone disease) is a disorder of connective tissues caused by mutations in the collagen genes. We previously showed that intrauterine transplantation of human blood fetal stem/stromal cells in OI mice (oim) resulted in a significant reduction of bone fracture. This work examines the cellular mechanisms and mechanical bone modifications underlying these therapeutic effects, particularly examining the direct effects of donor collagen expression on bone material properties. In this study, we found an 84% reduction in femoral fractures in transplanted oim mice. Fetal blood stem/stromal cells engrafted in bones, differentiated into mature osteoblasts, expressed osteocalcin, and produced COL1a2 protein, which is absent in oim mice. The presence of normal collagen decreased hydroxyproline content in bones, altered the apatite crystal structure, increased the bone matrix stiffness, and reduced bone brittleness. In conclusion, expression of normal collagen from mature osteoblast of donor origin significantly decreased bone brittleness by improving the mechanical integrity of the bone at the molecular, tissue, and whole bone levels.

The effects of fetal allogeneic umbilical cord tissue transplant following experimental spinal cord injury on urinary bladder morphology.

BACKGROUND AND PURPOSE: In continuation of our previous experimental study on spinal cord injury (SCI) using fetal stem cells, we investigated here the effects of fetal allogeneic umbilical cord tissue transplant on the urinary bladder morphology in a rat SCI model. MATERIAL AND METHODS: Five pregnant albino Wistar rats at 12 days of gestation were used to obtain the umbilical cord cell graft. In Group 1 (n = 5), Th8-Th9 laminectomy was performed. Group 2 (n = 5) received spinal cord injury. In Group 3 (n = 5), the cultured fetal umbilical cord cells coated with alginate gel were placed into the lesion cavity. In Group 4 (n = 5), only alginate sponges without umbilical cord cells were placed into the injury cavity. The bladders of animals were analyzed pathologically at 21 days after surgery. RESULTS: The thickness of the epithelium and the lamina propria did not differ among studied groups (p > 0.05). The lamina muscularis thickness was significantly higher in Group 2 and Group 4 than the others (p < 0.05). The bladder weight was similar among Groups 1, 2, and 3 (p > 0.05). Fibrosis was significantly increased in Group 2 (p < 0.05); it was greater in Group 2 than in Group 3 (p < 0.05) but did not differ between Groups 1 and 3 (p > 0.05). CONCLUSIONS: This study suggests that allogeneic umbilical cord tissue transplantation after SCI may prevent bladder wall hypertrophy and fibrosis in the rat SCI model.

Activin A, p15INK4b signaling, and cell competition promote stem/progenitor cell repopulation of livers in aging rats.

BACKGROUND & AIMS: Highly proliferative fetal liver stem/progenitor cells (FLSPCs) repopulate livers of normal recipients by cell competition. We investigated the mechanisms by which FLSPCs repopulate livers of older compared with younger rats. METHODS: Fetal liver cells were transplanted from DPPIV(+) F344 rats into DPPIV(-) rats of different ages (2, 6, 14, or 18 months); liver tissues were analyzed 6 months later. Cultured cells and liver tissues were analyzed by reverse transcription polymerase chain reaction, immunoblot, histochemistry, laser-capture microscopy, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling analyses. RESULTS: We observed 4- to 5-fold increases in liver repopulation when FLSPCs were transplanted into older compared with younger rats. Messenger RNA levels of cyclin-dependent kinase inhibitors increased progressively in livers of older rats; hepatocytes from 20-month-old rats had 6.1-fold higher expression of p15INK4b and were less proliferative in vitro than hepatocytes from 2-month-old rats. Expression of p15INK4b in cultured hepatocytes was up-regulated by activin A, which increased in liver during aging. Activin A inhibited proliferation of adult hepatocytes, whereas FLSPCs were unresponsive because they had reduced expression of activin receptors (eg, ALK-4). In vivo, expanding cell clusters derived from transplanted FLSPCs had lower levels of ALK-4 and p15INK4b and increased levels of Ki-67 compared with the host parenchyma. Liver tissue of older rats had 3-fold more apoptotic cells than that of younger rats. CONCLUSIONS: FLSPCs, resistant to activin A signaling, repopulate livers of older rats; hepatocytes in older rats have less proliferation because of increased activin A and p15INK4b levels and increased apoptosis than younger rats. These factors and cell types might be manipulated to improve liver cell transplantation strategies in patients with liver diseases in which activin A levels are increased.

Tg737 inhibition results in malignant transformation in fetal liver stem/progenitor cells by promoting cell-cycle progression and differentiation arrest.

Cancer stem/progenitor cells (CSPCs) may originate from the malignant transformation of normal stem cells. However, the mechanism by which normal stem cells undergo such transformation is not understood. Our previous studies provided evidence that Tg737 may play an important role in carcinogenesis of liver stem cells. In this study, we investigated the role of Tg737 in the malignant transformation of fetal liver stem/progenitor cells (FLSPCs). We inhibited Tg737 in FLSPCs using short hairpin RNA (shRNA). The microscopic observations of freshly purified Tg737 normal FLSPCs (nFLSPCs) and Tg737-silent FLSPCs (sFLSPCs), which showed high expression levels of stem cell markers, revealed no significant morphological changes in sFLSPCs. Following RNAi of Tg737, the mRNA and protein levels of sFLSPCs decreased by 81.81% and 80.10% as shown by PCR, Western blot and immunocytochemistry analyses. Excluding apoptosis-related effects, we found that silencing of Tg737 resulted in enhanced cell proliferation through promoting cell-cycle progression via upregulation of cyclin D1 and cyclin B expression (P < 0.05). Silencing of Tg737 also resulted in significant arrest of cell differentiation (P < 0.05), stable expression of both albumin (ALB) and alpha fetoprotein (AFP) (P > 0.05) and quiescent ultrastructure. Assessment of cell malignant traits by transwell migration assays and by growth of xenograft tumors in athymic mice showed that reduced expression of Tg737 greatly promoted cell invasion and hepatocarcinogenesis of FLSPCs (P < 0.05). This work shows that inactivation of Tg737 may play an important role in malignant transformation of FLSPCs.