Zhijun Yang

Neural differentiation ability of mesenchymal stromal cells from bone marrow and adipose tissue: a comparative study

BACKGROUND AIMS The characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual. METHODS The proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal. RESULTS The ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction. CONCLUSIONS Our study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases.

Transplantation of autologous bone marrow mesenchymal stem cells in the treatment of complete and chronic cervical spinal cord injury.

Neuronal injuries have been a challenging problem for treatment, especially in the case of complete and chronic cervical spinal cord injury (SCI). Recently, particular attention is paid to the potential of stem cell in treating SCI, but there are only few clinical studies and insufficient data. This study explored the efficacy of autologous bone marrow mesenchymal stem cells (BMMSCs) transplantation in the treatment of SCI. Forty patients with complete and chronic cervical SCI were selected and randomly assigned to one of the two experimental groups, treatment group and control group. The treatment group received BMMSCs transplantation to the area surrounding injury, while the control group was not treated with any cell transplantation. Both the transplant recipients and the control group were followed up to 6 months, postoperatively. Preoperative and postoperative neurological functions were evaluated with AIS grading, ASIA score, residual urine volume and neurophysiological examination. Results showed that in the treatment group 10 patients had a significant clinical improvement in terms of motor, light touch, pin prick sensory and residual urine volume, while nine patients showed changes in AIS grade. Neurophysiological examination was consistent with clinical observations. No sign of tumor was evident until 6 months postoperatively. In the control group, no improvement was observed in any of the neurological functions specified above. BMMSCs transplantation improves neurological function in patients with complete and chronic cervical SCI, providing valuable information on applications of BMMSCs for the treatment of SCI.

Tumourigenicity and Immunogenicity of Induced Neural Stem Cell Grafts Versus Induced Pluripotent Stem Cell Grafts in Syngeneic Mouse Brain

Along with the development of stem cell-based therapies for central nervous system (CNS) disease, the safety of stem cell grafts in the CNS, such as induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs), should be of primary concern. To provide scientific basis for evaluating the safety of these stem cells, we determined their tumourigenicity and immunogenicity in syngeneic mouse brain. Both iPSCs and embryonic stem cells (ESCs) were able to form tumours in the mouse brain, leading to tissue destruction along with immune cell infiltration. In contrast, no evidence of tumour formation, brain injury or immune rejection was observed with iNSCs, neural stem cells (NSCs) or mesenchymal stem cells (MSCs). With the help of gene ontology (GO) enrichment analysis, we detected significantly elevated levels of chemokines in the brain tissue and serum of mice that developed tumours after ESC or iPSC transplantation. Moreover, we also investigated the interactions between chemokines and NF-κB signalling and found that NF-κB activation was positively correlated with the constantly rising levels of chemokines, and vice versa. In short, iNSC grafts, which lacked any resulting tumourigenicity or immunogenicity, are safer than iPSC grafts.

Induced neural stem cells modulate microglia activation states via CXCL12/CXCR4 signaling

We previously reported that induced neural stem cells (iNSCs) directly reprogrammed from mouse embryonic fibroblasts can expand and differentiate into neurons, astrocytes and oligodendrocytes. Whether iNSCs have immunoregulatory properties in addition to facilitating cell replacement remains uncertain. In this study, we aimed to characterize the immunomodulatory effects of iNSCs on the activation states of microglia and to elucidate the mechanisms underlying these effects. Using a mouse model of closed head injury (CHI), we observed that iNSC grafts decreased the levels of ED1+/Iba1+ and TNF-α+/Iba1+ microglia but increased the levels of IGF1+/Iba1+ microglia in the injured cortex. Subsequently, using a Transwell co-culture system, we discovered that iNSCs could modulate LPS-pretreated microglia phenotypes in vitro via CXCL12/CXCR4 signaling, which we demonstrated through the administration of the CXCR4 antagonist AMD3100 and CXCR4-specific siRNA treatment. An in vivo loss-of-function study also revealed that iNSC grafts regulated the behavior of resident microglia via CXCL12/CXCR4 signaling, influencing their activation state such that they promoted neurological functional recovery and neuron survival. Furthermore, the beneficial effects of iNSC transplantation were significantly diminished by CXCR4 knockdown. In short, iNSCs have the potential to influence microglia activation and the acquisition of neuroprotective phenotypes via CXCL12/CXCR4 signaling.

Differentiation of cryopreserved human umbilical cord blood-derived stromal cells into cells with an oligodendrocyte phenotype

In this study, we examined the phenotypic characteristics of human umbilical cord blood-derived mesenchymal stromal cells (UCB-derived MSCs) differentiated along an oligodendrocyte pathway. We induced human UCB-derived MSCs to form floating neurospheres, and these neurospheres were then induced to differentiate into oligodendrocyte progenitor-like cells using multiple induction factors. Differentiated UCB-derived MSCs showed morphologic characteristics of an oligodendrocyte phenotype. The expression of cell surface markers characteristic of oligodendrocyte progenitor cells or oligodendrocytes was determined by immunocytochemical staining. These results suggest that human UCB-derived MSCs can be induced to differentiate into cells with an oligodendrocyte phenotype and that these cells may have potential in the future cellular therapy of central neurological disorders.

Systemic Administration of Induced Neural Stem Cells Regulates Complement Activation in Mouse Closed Head Injury Models

Complement activation plays important roles in the pathogenesis of central nervous system (CNS) diseases. Patients face neurological disorders due to the development of complement activation, which contributes to cell apoptosis, brain edema, blood-brain barrier dysfunction and inflammatory infiltration. We previously reported that induced neural stem cells (iNSCs) can promote neurological functional recovery in closed head injury (CHI) animals. Remarkably, we discovered that local iNSC grafts have the potential to modulate CNS inflammation post-CHI. In this study, we aimed to explore the role of systemically delivered iNSCs in complement activation following CNS injury. Our data showed that iNSC grafts decreased the levels of sera C3a and C5a and down-regulated the expression of C3d, C9, active Caspase-3 and Bax in the brain, kidney and lung tissues of CHI mice. Furthermore, iNSC grafts decreased the levels of C3d+/NeuN+, C5b-9+/NeuN+, C3d+/Map2+ and C5b-9+/Map2+ neurons in the injured cortices of CHI mice. Subsequently, we explored the mechanisms underlying these effects. With flow cytometry analysis, we observed a dramatic increase in complement receptor type 1-related protein y (Crry) expression in iNSCs after CHI mouse serum treatment. Moreover, both in vitro and in vivo loss-of-function studies revealed that iNSCs could modulate complement activation via Crry expression.

Neurotrophy and immunomodulation of induced neural stem cell grafts in a mouse model of closed head injury

Closed head injury (CHI) usually results in severe and permanent neurological impairments, which are caused by several intertwined phenomena, such as cerebral edema, blood-brain barrier (BBB) disruption, neuronal loss, astroglial scarring and inflammation. We previously reported that induced neural stem cells (iNSCs), similar to neural stem cells (NSCs), can accelerate neurological recovery in vivo and produce neurotrophic factors in vitro. However, the effects of iNSC neurotrophy following CHI were not determined. Moreover, whether iNSCs have immunomodulatory properties is unknown. Mouse models of CHI were established using a standardized weight-drop device and assessed by neurological severity score (NSS). Although these models fail to mimic the complete spectrum of human CHI, they reproduce impairment in neurological function observed in clinical patients. Syngeneic iNSCs or NSCs were separately transplanted into the brains of CHI mice at 12h after CHI. Neurological impairment post-CHI was evaluated by several tests. Animals were sacrificed for morphological and molecular biological analyses. We discovered that iNSC administration promoted neurological functional recovery in CHI mice and reduced cerebral edema, BBB disruption, cell death and astroglial scarring following trauma. Implanted iNSCs could up-regulate brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF) levels to support the survival of existing neurons after CHI. In addition, engrafted iNSCs decreased immune cell recruitment and pro-inflammatory cytokine expression in the brain post-injury. Moreover, we found significant nuclear factor-kappaB (NF-κB) inhibition in the presence of iNSC grafts. In short, iNSCs exert neurotrophic and immunomodulatory effects that mitigate CHI-induced neurological impairment.

Induced neural stem cell-derived astrocytes modulate complement activation and mediate neuroprotection following closed head injury

The complement system is a crucial component of immunity, and its activation has critical roles in neuroinflammatory response and cellular damage following closed head injury (CHI). We previously demonstrated that systemically injected induced neural stem cells (iNSCs) could modulate complement activation to ameliorate neuronal apoptosis in mouse CHI models. However, it remains unknown whether iNSC derivatives can regulate complement activation. In the present study, after CHI mouse serum treatment, we found dramatic decreases in the cellular viabilities of differentiated iNSCs. Interestingly, following CHI mouse serum treatment, the death of astrocytes derived from iNSCs which were pre-treated with CHI mouse serum was significantly decreased. Meanwhile, the deposition of C3 (C3d) and C5b-9 in these astrocytes was substantially reduced. Remarkably, we detected increased expression of complement receptor type 1-related protein y (Crry) in these astrocytes. Moreover, these astrocytes could reduce the numbers of apoptotic neurons via Crry expression post-CHI mouse serum treatment. Additionally, intracerebral-transplanted iNSCs, pre-treated with CHI mouse serum, significantly increased the levels of Crry expression in astrocytes to reduce the accumulation of C3d and C9 and the death of neurons in the brains of CHI mice. In summary, iNSCs receiving CHI mouse serum pre-treatment could enhance the expression of Crry in iNSC-derived astrocytes to modulate complement activation and mediate neuroprotection following CHI.

Syringe needle skull penetration reduces brain injuries and secondary inflammation following intracerebral neural stem cell transplantation

Intracerebral neural stem cell (NSC) transplantation is beneficial for delivering stem cell grafts effectively, however, this approach may subsequently result in brain injury and secondary inflammation. To reduce the risk of promoting brain injury and secondary inflammation, two methods were compared in the present study. Murine skulls were penetrated using a drill on the left side and a syringe needle on the right. Mice were randomly divided into three groups (n=84/group): Group A, receiving NSCs in the left hemisphere and PBS in the right; group B, receiving NSCs in the right hemisphere and PBS in the left; and group C, receiving equal NSCs in both hemispheres. Murine brains were stained for morphological analysis and subsequent evaluation of infiltrated immune cells. ELISA was performed to detect neurotrophic and immunomodulatory factors in the brain. The findings indicated that brain injury and secondary inflammation in the left hemisphere were more severe than those in the right hemisphere, following NSC transplantation. In contrast to the left hemisphere, more neurotrophic factors but less pro-inflammatory cytokines were detected in the right hemisphere. In addition, increased levels of neurotrophic factors and interleukin (IL)-10 were observed in the NSC transplantation side when compared with the PBS-treated hemispheres, although lower levels of IL-6 and tumor necrosis factor-α were detected. In conclusion, the present study indicated that syringe needle skull penetration vs. drill penetration is an improved method that reduces the risk of brain injury and secondary inflammation following intracerebral NSC transplantation. Furthermore, NSCs have the potential to modulate inflammation secondary to brain injuries.