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Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy., 遺伝子治療によるホスト脳の環境最適化が細胞移植効果を高める --ホスト脳へのL1CAMの強制発現によるマウス胎仔脳移植片の軸索伸長促進効果--. 京都大学プレスリリース. 2023-03-24., Combining cell transplantation and gene therapy to enhance axonal outgrowth in the central nervous system. 京都大学プレスリリース. 2023-04-06.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

Abstract 4690 Hypophosphatasia (HPP) is one of the bone metabolic disorders caused by mutations of the liver/bone/kidney alkaline phosphatase (ALPL) gene encoding tissue-nonspecific alkaline phosphatase (ALP). The disease is characterized by the disturbance of bone and tooth mineralization and reduced serum ALP activity. Clinical severity of HPP often depends on the age of onset. Patients with perinatal and infantile forms usually have poor prognosis. Phenotype in patients with HPP is also closely related to residual enzyme activity affected by ALPL mutations. Most perinatal patients die of respiratory failure resulting from the reduced osteogenesis of the thorax, suggesting that production of new bones that support respiration in the chest would improve the prognosis. However, despite extensive studies on the molecular pathogenesis of HPP, there is no curative treatment for HPP. Mesenchymal stem cells (MSCs), which normally reside in bone marrow (BM), umbilical cord blood, muscle connective tissue, and adipose tissue, have multipotency that differentiate into various mesenchymal lineages and several mesoderm lineages. These include bone, cartilage, tendon, muscle, e adipose tissue, neuron, hepatocyte, and endothelium. MSC aloso modulates immune system and hematopoietic stem cell fate. These miscellaneous properties of MSCs are clinically applied such as prophylaxis for graft versus host disease (GVHD) or regeneration of ischemic heart because MSCs can easily be isolated and expanded in vitro while maintaining genetic stability. However, MSCs have never been used to facilitate bone formation or osteogenesis in patients with severe HPP. Herein we performed transplantation of MSCs expanded ex vivo for patients with severe HPP that underwent preceding BM transplantation (BMT) to determine the effects of MSCs on new bone generation. Two patients with severe hypophosphatasia were studied. Both patients were diagnosed as fatal HPP based on perinatal onset, respiratory disturbance, and ALPL mutations with extremely low ALP activity. MCSs obtained from these patients had little osteogenic activity in vitro. Their donors were asymptomatic relatives of the patients harboring heterozygous mutation of the ALPL gene. Osteogenic activity of donor-MSCs was normal. Myeloablative conditioning regimen was used for BMT. MSCs obtained from BM were expanded ex vivo in a flask containing a-minimum essential medium with 15% fetal bovine serum and 10 mg/mL kanamycin sulfate. The adherent cells became nearly confluent after 10 days and the expression of the cell surface antigens was mesenchymal type (CD34-, CD45-, CD105+, and SH3+). Case1: An 8-month-old girl was administered with buslfan (BU), fludarabine, and antithymocyte globulin (ATG). Tacrolimus, mycophenolate mofetil, and a short course of methotrexate (sMTX) were used for to GVHD prophylaxis. BMT was performed using marrow cells derived from a haploidentical donor. Cultured MSCs with cultured osteoblasts were administered 1 month after BMT. Although donor BM did not engraft, her respiratory condition was improved. Secondary MSCs transplantation (MSCT) was performed when her respiratory condition became deteriorated, which led to a significant improvement in her respiratory condition, her limbs length, and her physical development. Moreover, FISH and PCR analyses revealed that de novo formation of bone derived from both donor and recipient cells. Although, unexpectedly, she developed chronic myeloid leukemia with BCR-ABL fusions 2 years after BMT, she remained remission of the disease after secondary BMT from same donor. Mineralization of metaphysis improved although that of other bones such as long bones did not. Case2: A 14-month-old boy underwent BMT from a HLA 2 locus mismatched related donor using BU, cyclophosphamide, and ATG. Tacrolimus and sMTX were used for GVHD prophylaxis. Ex vivo expanded MSCs were transplanted 2 weeks after BMT. Engraftment of donor derived BM was confirmed. His condition gradually improved. Our data suggests that combination of MSCT and BMT may offer novel and effective treatment modalities to facilitate osteogenesis in patients with fatal HPP. Disclosures: No relevant conflicts of interest to declare.