Toshiaki Numajiri

Adipose-Derived Stem Cells Produce Factors Enhancing Peripheral Nerve Regeneration: Influence of Age and Anatomic Site of Origin

Adipose-derived stem cells (ADSCs) are attracting increased attention as a novel source in regenerative medicine. Transplantation of ADSCs promotes functional recovery in animal models of peripheral nerve injury, but the mechanism of enhanced nerve regeneration remains to be elucidated. In addition, it is important to examine whether the supportive functions of ADSCs are dependent on donor age or anatomic site of origin. In this study, we examined the effects of factors produced by mouse ADSCs on Schwann cells (SCs) and dorsal root ganglion (DRG) neurons in vitro and compared these effects among ADSCs from donors of different age and from different anatomic regions. ADSC-derived soluble factors supported survival and proliferation of SCs and promoted neurite outgrowth in DRG neurons. These beneficial effects were far superior to that of factors from 3T3-L1 cells and comparable to those of SC- and astrocyte (AC)-derived factors. ADSCs from different sources similarly retained their neurotrophic activity. Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay analyses demonstrated that ADSCs produced various growth factors, some of which were more abundant than in SCs and ACs. These results suggest that ADSCs promote peripheral nerve regeneration partly through paracrine secretion of trophic factors and regardless of donor age or anatomic site of origin.

Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

Recent studies have shown that adipose-derived stromal/stem cells (ASCs) contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs). This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2) and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta). NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.

Adipose-Derived Stem Cells Promote Peripheral Nerve Regeneration In Vivo without Differentiation into Schwann-Like Lineage.

Background: During recent decades, multipotent stem cells were found to reside in the adipose tissue, and these adipose-derived stem cells were shown to play beneficial roles, like those of Schwann cells, in peripheral nerve regeneration. However, it has not been well established whether adipose-derived stem cells offer beneficial effects to peripheral nerve injuries in vivo as Schwann cells do. Furthermore, the in situ survival and differentiation of adipose-derived stem cells after transplantation at the injured peripheral nerve tissue remain to be fully elucidated. Methods: Adipose-derived stem cells and Schwann cells were transplanted with gelatin hydrogel tubes at the artificially blunted sciatic nerve lesion in mice. Neuroregenerative abilities of them were comparably estimated. Cre-loxP–mediated fate tracking was performed to visualize survival in vivo of transplanted adipose-derived stem cells and to investigate whether they differentiated into Schwann linage cells at the peripheral nerve injury site. Results: The transplantation of adipose-derived stem cells promoted regeneration of axons, formation of myelin, and restoration of denervation muscle atrophy to levels comparable to those achieved by Schwann cell transplantation. The adipose-derived stem cells survived for at least 4 weeks after transplantation without differentiating into Schwann cells. Conclusions: Transplanted adipose-derived stem cells did not differentiate into Schwann cells but promoted peripheral nerve regeneration at the injured site. The neuroregenerative ability was comparable to that of Schwann cells. Adipose-derived stem cells at an undifferentiated stage may be used as an alternative cell source for autologous cell therapy for patients with peripheral nerve injury.

Tracheal reconstruction with a prefabricated and double-folded radial forearm free flap

We have described a prefabricated radial forearm free flap containing costal cartilage for tracheal reconstruction. The main advantages of this flap are that it requires only one piece of costal cartilage, simple cartilage trimming, and allows the possible reconstruction of both the internal lining and the external resurfacing when the flap itself is folded. This procedure is easy and its outcome is stable. This flap is an attractive option for the reconstruction of large tracheal and skin defects, even in areas previously exposed to surgery or irradiation.

Double vascular anastomosis in the neck for reliable free jejunal transfer

To reduce the possibility of necrosis in free jejunal transfer after total pharyngolaryngo-oesophagectomy, we made a second set of arterial and venous anastomoses in addition to the usual cervical arterial and venous anastomoses. To obtain two different arterial flows (carotid and subclavian systems), the recipient arteries were the superior thyroid and the transverse cervical. For venous flow, the internal and external jugular veins were used. All flaps survived. This procedure, which is limited to the already dissected neck with no procedures in other areas, is less invasive than others. Its advantages are that when one vessel has thrombosed the other vessel functions as a safety valve, and the greater pliability of the vascular pedicles makes it easier to reach the recipient vessels. Because this method is theoretically safe and results in less possibility of losing a flap when the anastomosis is unreliable, the double-pedicled transfer can be an option for safer free jejunal transfer.

Double vascular anastomosis for safer free jejunal transfer in unfavorable conditions.

Free jejunum was transferred to a patient with recurrent hypopharyngeal carcinoma under unfavorable cervical conditions, caused by prior therapeutic chemoradiotherapy for hypopharyngeal carcinoma and gastric pull-up with cervical leak, resulting from treatment for thoracic esophageal cancer. The cervical recipient vessels were buried in extensively scarred fibrous tissues, so they were thought to be less reliable. Because postoperative vascular occlusion was anticipated, in addition to the ordinary single vascular anastomosis to the damaged cervical vessels, secondary vascular anastomosis to the healthy chest vessels was performed. We designed the graft to have double vascular pedicles that communicated with each other through arcade vessels. This made it possible to anastomose doubly to an intact thoracoacromial artery in the chest by elongating the vascular pedicles of the mesentery without the need for an interpositional vein graft, in addition to ordinary anastomosis in the damaged neck. The flap is nourished by the vessels from two different origins (carotid and axillary arteries, internal jugular and axillary veins) at two different places (damaged and healthy areas). This method increases the quantity of feeding vessels while improving the quality of the recipient vessels within the local area and flow sources. It is one treatment option when conditions are unfavorable for safer jejunal transfer.

Does a vascular supercharge improve the clinical outcome for free jejunal transfer

To clarify whether a supercharged free jejunal transfer would have a different clinical outcome from the usual transfer method, we examined clinical data from cases of esophago‐pharyngeal reconstruction. Fifty‐three patients in whom the hypopharynx and cervical esophagus was reconstructed with a free jejunal transfer were divided into two groups: 19 normal procedures and 34 supercharged. Clinical outcomes including intraoperative and postoperative events, complications and deglutition were compared statistically. There were no significant differences between the groups in terms of the rates of free flap failure, leakage, stenosis, drinking status, dysphagia, or operating time. There were no significant advantages in clinical outcomes when using a supercharge. However, supercharged flaps with an intraoperative arterial thrombosis were all rescued and survived. Thus, a supercharge in free flap is not necessary for all cases. Its indication should be limited to cases when free flaps are not reliable because of intraoperative thrombosis and arterial insufficiency.

Low-cost Design and Manufacturing of Surgical Guides for Mandibular Reconstruction Using a Fibula.

Background: Surgical cutting guides are used in mandibular reconstruction involving osteotomy of the mandible and fibula. Cutting guides produced using computer-aided design (CAD) and computer-aided manufacturing (CAM) technologies have been reported recently. These guides aim to increase the benefits to patients by improving the accuracy, shortening the operating time, and correcting occlusion. However, the availability of these advanced technologies is limited in some regions of the world. To test whether we could produce low-cost surgical cutting guides, we made surgical guides and investigated their accuracy. Methods: Using free CAD software, we designed surgical cutting guides for the mandible and fibula and used these to perform virtual mandibular segmental osteotomies and fibula transplants in 12 model surgeries. The cutting guides were printed on a 3-dimensional (3D) printer. The model surgeries were performed using 3D mandibular models and cutting guides to check their accuracy. Deviations between the virtually simulated plan and the actual model surgery were investigated. Results: CAD and CAM technologies were used to design and 3D print the cutting guides and models. The guided surgeries were performed. The deviations were about 1.3 mm for mandibular osteotomy, less than 1 mm for fibular osteotomy, and within 2.4 mm for reconstructions of the mandible. Conclusions: Without using expensive software or products, we were able to design surgical cutting guides for the mandible and fibula and used these to perform virtual simulation of mandibular segmental osteotomy and fibular reconstruction. Model surgeries using 3D-printed surgical guides showed that the accuracy of reconstruction was within a 3-mm deviation. In circumstances where commercial CAD/CAM guides are not available, it may be possible to use CAD/CAM surgical guides in the clinic if doctors are willing to volunteer their time for the design and printing.

Artery-dominant free jejunal transfer

Although the supercharge (additional microvascular anastomosis) technique is often used in pedicled transfer of parts of the gastrointestinal tract, this is rarely performed during free jejunal transfer (FJT). The differences in blood circulation and outcomes between the usual single pedicle flap and a double pedicle flap are not well known. Therefore, we evaluated the effect of an additional arterial anastomosis in FJT. The FJT was performed using one venous and two arterial anastomoses after hypopharyngeal cancer ablation. To assess the effects of an arterial supercharge, blood-gas analysis, including the venous partial pressure of oxygen (pO(2)) and partial pressure of carbon dioxide (pCO(2)), was performed on samples drawn thrice from the jejunal vein: before harvest, after the anastomosis of a paired artery and vein and after an additional arterial anastomosis. The result revealed that the venous pO(2) was elevated by the additional arterial anastomosis, compared with the two other measuring times (P=0.04). The venous pCO(2) did not show significant changes. By being given a dominant artery, a jejunal flap can develop a physiological circulatory environment and can establish nutritional pathways without adverse effects.

Direct Conversion of Human Fibroblasts into Schwann Cells that Facilitate Regeneration of Injured Peripheral Nerve In Vivo

Schwann cells (SCs) play pivotal roles in the maintenance and regeneration of the peripheral nervous system. Although transplantation of SCs enhances repair of experimentally damaged peripheral and central nerve tissues, it is difficult to prepare a sufficient number of functional SCs for transplantation therapy without causing adverse events for the donor. Here, we generated functional SCs by somatic cell reprogramming procedures and demonstrated their capability to promote peripheral nerve regeneration. Normal human fibroblasts were phenotypically converted into SCs by transducing SOX10 and Krox20 genes followed by culturing for 10 days resulting in approximately 43% directly converted Schwann cells (dSCs). The dSCs expressed SC‐specific proteins, secreted neurotrophic factors, and induced neuronal cells to extend neurites. The dSCs also displayed myelin‐forming capability both in vitro and in vivo. Moreover, transplantation of the dSCs into the transected sciatic nerve in mice resulted in significantly accelerated regeneration of the nerve and in improved motor function at a level comparable to that with transplantation of the SCs obtained from a peripheral nerve. The dSCs induced by our procedure may be applicable for novel regeneration therapy for not only peripheral nerve injury but also for central nerve damage and for neurodegenerative disorders related to SC dysfunction. Stem Cells Translational Medicine 2017;6:1207–1216