C. Aguayo

Functional recovery of chronic paraplegic pigs after autologous transplantation of bone marrow stromal cells.

Background. Bone marrow stromal cells (BMSC) transplantation offers promise in the treatment of chronic paraplegia in rodents. Here, we report the effect of this cell therapy in adult pigs suffering chronic paraplegia. Methods. Three months after spinal cord injury, autologous BMSC in autologous plasma was injected into lesion zone and adjacent subarachnoid space in seven paraplegic pigs. On the contrary, three paraplegic pigs only received autologous plasma. Functional outcome was measured weekly until the end of the follow-up, 3 months later. Results. Our present study showed progressive functional recovery in transplanted pigs. At this time, intramedullary postraumatic cavities were filled by a neoformed tissue containing several axons, together with BMSC that expressed neuronal or glial markers. Furthermore, in the treated animals, electrophysiological studies showed recovery of the previously abolished somatosensory-evoked potentials. Conclusions. These findings confirm previous observations in rodents and support the possible utility of BMSC transplantation in humans suffering chronic paraplegia.

Delayed intralesional transplantation of bone marrow stromal cells increases endogenous neurogenesis and promotes functional recovery after severe traumatic brain injury.

Primary objective: To investigate the utility of delayed transplantation of bone marrow stromal cells (BMSC) to improve the neurological sequels after traumatic brain injury (TBI). Methods: Adult Wistar rats were subjected to weight-drop impact causing severe brain injury, and 2 months later, BMSC in saline, or saline alone, were injected into injured brain tissue. Both experimental groups were evaluated by means of rotarod and modified neurologic severity scores (mNSS) tests in the course of the two following months. At this time, the animal were sacrificed and their brains were studied by means of histological and immunohistochemical techniques. Results: Rotarod and mNSS tests showed progressive functional recovery in the BMSC- transplanted rats, compared with controls. Two months after transplantation, BMSC survived in the host tissue, and some of them showed expression of Neu-N or GFAP, suggesting neuronal and astroglial transdifferentiation. Furthermore, significant increase of endogenous neurogenesis was found in BMSC-transplanted rats, compared with controls. Conclusions: These findings suggest the utility of delayed intracerebral transplantation of BMSC for the treatment of established sequels after TBI.

Cell therapy for spinal cord repair: optimization of biologic scaffolds for survival and neural differentiation of human bone marrow stromal cells

BACKGROUND AIMS The suppression of cell apoptosis using a biodegradable scaffold to replace the missing or altered extracellular matrix (ECM) could increase the survival of transplanted cells and thus increase the effectiveness of cell therapy. METHODS We studied the best conditions for the proliferation and differentiation of human bone marrow stromal cells (hBMSC) when cultured on different biologic scaffolds derived from fibrin and blood plasma, and analyzed the best concentrations of fibrinogen, thrombin and calcium chloride for favoring cell survival. The induction of neural differentiation of hBMSC was done by adding to these scaffolds different growth factors, such as nerve growth factor (NGF), brain-derived-neurotrophic factor (BDNF) and retinoic acid (RA), at concentrations of 100 ng/mL (NGF and BDNF) and 1 micro/mL (RA), over 7 days. RESULTS Although both types of scaffold allowed survival and neural differentiation of hBMSC, the results showed a clear superiority of platelet-rich plasma (PRP) scaffolds, mainly after BDNF administration, allowing most of the hBMSC to survive and differentiate into a neural phenotype. CONCLUSIONS Given that clinical trials for spinal cord injury using hBMSC are starting, these findings may have important clinical applications.

Neural transdifferentiation of bone marrow stromal cells obtained by chemical agents is a short-time reversible phenomenon

Bone marrow stromal cells (BMSC) can acquire morphological and immunohistochemical features of neural cells when they are treated with diverse chemical agents, a finding interpreted as result of cell transdifferentiation. With the purpose of a better knowledge of the possible utility of BMSC for strategies of Nervous System (NS) repair, we have studied the morphological and immunohistochemical changes induced in BMSC by chemical agents, in comparison with those that happen when BMSC are co-cultured with Schwann cells. While chemical BMSC transdifferentiation is a short-time reversible phenomenon, BMSC transdifferentiation obtained by Schwann cell-derived neurotrophic factors remains stable after it has been reached. These findings question the possible clinical utility of BMSC trandifferentiation using chemical agents, and support that neural transdifferentiation of BMSC is a biological phenomenon that can be obtained in vivo because of the presence of environmental factors.

Neurotrophic Schwann-cell factors induce neural differentiation of bone marrow stromal cells.

Neural transdifferentiation of bone marrow stromal cells has been questioned, because cell fusion could explain the development of new cell types, misinterpreted as transdifferentiated cells. We performed here cocultures of bone marrow stromal cells and Schwann cells, without possibility that both cell types can establish contact. In these conditions, bone marrow stromal cells expressed nestin 4 h after beginning cocultures, and strong expression of neuronal markers was disclosed at 72 h, increasing at 1 and 2 weeks. Our results support that neural transdifferentiation of bone marrow stromal cells is induced by soluble factors provided by glial cells, and suggest that cell fusion should not be significant when local bone marrow stromal cells administration for neural repair is considered.

Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis

BACKGROUND AIMS When a severe neurologic lesion occurs as a consequence of intracerebral hemorrhage (ICH), there is no effective treatment available for improving the outcome. However, cell therapy has opened new perspectives on reducing neurologic sequels subsequent to this disease. METHODS In this study, ICH was induced by stereotactic injection of 0.5 U collagenase type IV in the striatum of adult Wistar rats, and 2 h later a group of animals (n = 48) was subjected to intracerebral injection of 2 × 10(6) allogeneic bone marrow stromal cells (BMSC), while a control group (n = 48) received saline only. Eight animals from each group were killed at 48 h, 72 h, 7 days, 14 days, 21 days and 28 days. At these time-points, endogenous neurogenesis and survival of transplanted BMSC were studied. RESULTS Our findings show that after allogeneic BMSC transplantation, donor cells can survive in the brain tissue expressing neuronal and astroglial markers. Furthermore, BMSC transplantation enhances endogenous neurogenesis and inhibits apoptosis of newborn neural cells. CONCLUSIONS Although these results should be extrapolated to human disease with caution, it is obvious that cell therapy using allogeneic BMSC transplantation offers great promise for developing novel and efficacious strategies in patients suffering ICH.

The pig model of chronic paraplegia: A challenge for experimental studies in spinal cord injury

The regenerative medicine techniques that are beginning to be applied to the nervous system have led to increased hope in the treatment of diseases that have been considered incurable and that require experimental models on which to test new therapeutic strategies. We present our experience with adult pigs (minipigs) that have undergone a traumatic spinal cord injury (SCI) experimental model, and that have been followed for 1 year. We describe the surgical aspects of our SCI model by acute compression and also describe protocols for daily care and rehabilitation that are necessary to maintain the paraplegic pigs in good health during the months following the injury. Furthermore, we provide in detail the main complications that arise with this experimental model and the treatments used to address these complications. Suitable housing conditions, daily rehabilitation and prevention of complications (i.e., taking the same care applied to patients following SCI) are essential for achieving the absence of mortality and long-term maintenance of the animals. We consider the model that is described here to be feasible and useful for preliminary testing of novel therapeutic strategies aimed at regeneration of the injured spinal cord in paraplegic patients.

Late transplantation of allogeneic bone marrow stromal cells improves neurologic deficits subsequent to intracerebral hemorrhage

BACKGROUND AIMS Stem cell therapy seems to be a promising therapeutic tool for treating central nervous system (CNS) injuries. Bone marrow stromal cell (BMSC) transplantation influences functional outcome subsequent to intracerebral hemorrhage (ICH), and enhances endogenous neurogenesis in acute condition studies. We investigated whether late administration of BMSC improves functional deficits subsequent to ICH. METHODS Experimental ICH was induced by stereotactic injection of 0.5 IU collagenase type IV in the striatum of adult female Wistar rats, and 2 months later intralesional administration of 5 × 10(6) allogeneic BMSC from male donors rats in saline (n = 10), or saline only (n = 10), was performed. In the following 6 months, functional outcome was evaluated in each animal by rotarod, modified neurologic severity score (mNSS) and video-tracking box (VTB) tests. To study the behavior of BMSC after transplantation, in situ hybridization studies were performed, with double labeling of the chromosome Y-linked SrY-gene, and neuronal nuclei (NeuN) protein or gliofibrillary acidic protein (GFAP). RESULTS The assessment test revealed significant improvements in functional outcome for the BMSC-treated animals after 2 months of follow-up. Histologic results showed that functional outcome was associated with strong reactivation of endogenous neurogenesis. Furthermore, intralesional BMSC not only integrated in the injured tissue but also showed phenotypic expression of GFAP and NeuN. CONCLUSIONS Late intracerebral transplantation of allogeneic BMSC induces functional recovery after ICH. The possibility of using this type of cell therapy to reverse the consequences of hemorrhagic stroke in humans should be considered.

An approach to personalized cell therapy in chronic complete paraplegia: The Puerta de Hierro phase I/II clinical trial.

BACKGROUND AIMS Cell transplantation in patients suffering spinal cord injury (SCI) is in its initial stages, but currently there is confusion about the results because of the disparity in the techniques used, the route of administration, and the criteria for selecting patients. METHODS We conducted a clinical trial involving 12 patients with complete and chronic paraplegia (average time of chronicity, 13.86 years; SD, 9.36). The characteristics of SCI in magnetic resonance imaging (MRI) were evaluated for a personalized local administration of expanded autologous bone marrow mesenchymal stromal cells (MSCs) supported in autologous plasma, with the number of MSCs ranging from 100 × 10(6) to 230 × 10(6). An additional 30 × 10(6) MSCs were administered 3 months later by lumbar puncture into the subarachnoid space. Outcomes were evaluated at 3, 6, 9 and 12 months after surgery through clinical, urodynamic, neurophysiological and neuroimaging studies. RESULTS Cell transplantation is a safe procedure. All patients experienced improvement, primarily in sensitivity and sphincter control. Infralesional motor activity, according to clinical and neurophysiological studies, was obtained by more than 50% of the patients. Decreases in spasms and spasticity, and improved sexual function were also common findings. Clinical improvement seems to be dose-dependent but was not influenced by the chronicity of the SCI. CONCLUSION Personalized cell therapy with MSCs is safe and leads to clear improvements in clinical aspects and quality of life for patients with complete and chronically established paraplegia.

Cell therapy with bone marrow stromal cells after intracerebral hemorrhage: impact of platelet-rich plasma scaffolds

BACKGROUND AIMS Cell therapy using bone marrow stromal cells (BMSCs) has been considered a promising strategy for neurologic sequelae after intracerebral hemorrhage (ICH). However, after intracerebral administration of BMSCs, most of the cells die, partly because of the absence of extracellular matrix. Intracerebral transplantation of BMSCs, supported in a platelet-rich plasma (PRP) scaffold, optimizes this type of cell therapy. METHODS ICH was induced by stereotactic injection of 0.5 IU of collagenase type IV in the striatum of adult Wistar rats (n = 40). Two months later, the rats were subjected to intracerebral administration of 5 × 10(6) allogeneic BMSCs embedded in a PRP scaffold (n = 10), 5 × 10(6) allogeneic BMSCs in saline (n = 10), PRP-derived scaffold only (n = 10) or saline only (n = 10). Functional improvements in each group over the next 6 months were assessed using Rotarod and Video-Tracking-Box tests. Endogenous neurogenesis and survival of transplanted BMSCs were examined at the end of follow-up. RESULTS Our study demonstrated neurologic improvement after BMSC transplantation and significantly better functional improvement for the group of animals that received BMSCs in the PRP-derived scaffold compared with the group that received BMSCs in saline. Histologic results showed that better functional outcome was associated with strong activation of endogenous neurogenesis. After intracerebral administration of BMSCs, donor cells were integrated in the injured tissue and showed phenotypic expression of glial fibrillary acidic protein and neuronal nucleus. CONCLUSIONS PRP-derived scaffolds increase the viability and biologic activity of BMSCs and optimize functional recovery when this type of cell therapy is applied after ICH.