Skeletal myoblast transplantation for attenuation of hyperglycaemia, hyperinsulinaemia and glucose intolerance in a mouse model of type 2 diabetes mellitus.

AIMS/HYPOTHESIS: We aimed to demonstrate the feasibility and efficacy of intra-muscular transplantation of human skeletal myoblasts (hSkMs) for attenuation of hyperglycaemia and improvement of insulin sensitivity using a mouse model of type 2 diabetes mellitus. METHODS: KK Cg-Ay/J mice, aged 12 to 14 weeks, underwent an initial intraperitoneal glucose tolerance test (GTT) and were divided into the following groups: KK control group, basal medium (M199) only; KK myoblast group, with hSkM transplantation; KK fibroblast group, with human fibroblast transplantation. Non-diabetic C57BL mice were used as an additional normal control and also had hSkM transplantation. Cells were transplanted intra-muscularly into the skeletal muscles of the mice. All animals were treated with ciclosporin for 6 weeks only. HbA(1c) and fasting GTT, as well as serum adiponectin, cholesterol, insulin and triacylglycerol were studied. RESULTS: Immunohistochemistry studies showed extensive survival of the transplanted hSkMs in the skeletal muscles at 12 weeks, with nuclei of the hSkMs integrated into the host muscle fibres. Repeat GTT showed a significant decrease in glucose concentrations in the KK myoblast group compared with the KK control and KK fibroblast groups. The KK myoblast group also had reduced mean HbA(1c), cholesterol, insulin and triacylglycerol, and increased adiponectin compared with the KK control and KK fibroblast groups. C57BL mice showed no change in glucose homeostasis after hSkM transplant. CONCLUSIONS/INTERPRETATION: Human skeletal myoblast transplantation attenuated hyperglycaemia and hyperinsulinaemia and improved glucose tolerance in the KK mouse. This novel approach of improving muscle insulin resistance may be a potential alternative treatment for type 2 diabetes mellitus.

Effectiveness of transient immunosuppression using cyclosporine for xenomyoblast transplantation for cardiac repair.

We studied the survival of human myoblast for cellular myocardial reconstruction in a porcine model of chronic myocardial ischemia with immune tolerance using transient immunosuppression. A porcine model of chronic cardiac ischemia was created in 10 pigs (DMEM medium-injected n = 4; myoblast transplanted n = 6) by clamping ameroid ring around left circumflex coronary artery. Three weeks later, 3 x 10(8) human myoblasts carrying lac-z reporter gene were transplanted in multiple sites (0.25 mL each) into the left ventricular wall. Immunosuppression was achieved with 5 mg/kg cyclosporine for 6 weeks after cell transplantation. After animals were euthanized between 6 and 30 weeks after cell transplantation; the heart was removed for histological studies. Discontinuation of immunosuppression after 6 weeks of cell transplantation did not result in donor cell rejection. The lac-z-positive donor cells were detected in porcine host cardiac tissue for up to 30 weeks posttransplantation, expressing human skeletal myosin heavy chain. The results highlight the effectiveness of transient immunosuppression for myoblast transplantation for cardiac repair.

Use of developmental language scales in Chinese children.

The Reynell Developmental Language Scale (RDLS) and Symbolic Play Test (SPT) have been useful language tests for assessing the language age of children. Both tests have been validated in English-speaking children. However, there have been no studies conducted for Chinese children, whether Mandarin (Northerners) or Cantonese (Southerners) is used as the main dialect. As the Chinese population is the largest ethnic group in the world, and Chinese emigration occurred to nearly all parts of the world, it is essential to test whether these language tools can be applied for this ethnic group. The objective of this research was to study whether RDLS and SPT are useful in assessing the language age of Chinese children. Both RDLS (Chinese version) and SPT are conducted for 116 Chinese (Cantonese-speaking) children, aged 13-59 months, in Hong Kong. There is a significant positive correlation of the language age using RDLS and SPT with the chronological age of Chinese children. Both RDLS and SPT can be adopted in determining the language/mental age of Chinese (Cantonese-speaking) children.

Interventional radiology. Application to family practice.

New minimally invasive interventional radiologic procedures are being developed and refined. These alternatives to standard surgical treatments have fewer complications, shorter hospital stays, and lower costs. A variety of procedures that assist in both benign and malignant diseases are particularly suited for palliative care in end-stage malignancy.

Cell transplantation as an experimental treatment for Duchenne muscular dystrophy.

The feasibility, safety, and efficacy of myoblast transfer therapy (MIT) were assessed in an experimental lower body treatment (LBT) involving 32 Duchenne muscular dystrophy (DMD) boys aged 6-14 yr, half of whom were nonambulatory. Through 48 injections, five billion (55.6 x 10(6)/mL) normal myoblasts were transferred into 22 major muscles in both lower limbs, in 10 min with the subject under general anesthesia. Ten subjects received myoblasts cultured from satellite cells derived from 1-g fresh muscle biopsies of normal males aged 9-21 yr. Donor myoblasts for the remaining 22 boys were subcultured from reserves frozen 1 mo-1.5 yr ago. Only four donors were known to have identical histocompatibility with their recipients. All subjects took oral doses of the immunosuppressant cyclosporine (Cy), beginning at 2 days before MTT and lasting for 6 mo after MTT to facilitate donor cell survival. There was no evidence of an adverse reaction to MTT or Cy as determined by serial laboratory evaluations including electrolytes, creatinine, and urea. Objective functional tests using the KinCom Robotic Dynamometer measured the maximum isometric contractile forces of the ankle plantar flexors (AF), knee flexors (KF), and knee extensors (KE) before MTT and at 3, 6, and 9 mo after MTT. The AF, being distal muscles and less degenerative than the KE and the KF, showed no decrease in mean contractile force 3 mo after MTT, and progressive increases in force at 6 and 9 mo after MTT. At 9 mo after MTT, 60% of the 60 AF examined showed a mean increase of 50% in force; 28% showed no change; and only 12% showed a mean decrease in force of 29% when compared to the function of the same muscles before MTT. The KF, being proximal muscles and more degenerative, showed no change in function at 9 mo after MTT. The KE, being proximal and anti-gravitational, were most degenerative before MTT. They showed no statistically significant change in force at 3 mo after MTT but showed decreases at 6 and 9 mo after MTT. At 9 mo after MTT, 23% of the 60 KE examined showed a mean increase of 65% in force; 22% showed no change; and 55% showed a mean decrease of 24% in force. When results of all muscle groups (AF, KF, KE) were pooled, there was no change in force at 3, 6, or 9 mo after MTT vs. before MTT according to the Wilcoxon signed rank test.(ABSTRACT TRUNCATED AT 400 WORDS)

Dystrophin cytochemistry in mdx mouse muscles injected with labeled normal myoblasts.

A new technique enables correlation of dystrophin expression with the location of donor versus host nuclei in the same sections of mdx mouse muscle injected with normal myoblasts. Myoblasts from C57BL/6J mice or from humans were labeled with 0.01% fluoro-gold (FG) in Dulbecco's Modified Eagles Medium (DMEM) for 16 h at 37 degrees C before myoblast transfer. About 3 x 10(4) myoblasts were injected into the quadriceps muscles of mdx mice immunosuppressed with cyclosporine A (CsA). At 11, 21, or 25 days after myoblast transfer, injected muscles were dissected out and sectioned. These mouse sections were processed for dystrophin and then labeled with a fluorescent nucleus counterstain, 5 micrograms% Hoechst 33342 in phosphate-buffered saline (PBS), for 10 min at room temperature. Fluoro-gold labeling corresponding with Hoechst 33342 staining indicated survival of normal nuclei in dystrophic muscle. Dystrophin was found in the sarcolemma of myofibers containing FG-labeled nuclei but not of myofibers containing only Hoechst 33342-labeled nuclei. Control muscle samples showed neither FG labeling nor dystrophin. This study demonstrates that the donor human and mouse myoblasts survived and developed in host mouse muscles for at least 25 days after myoblast transfer, and that the localization of their normal nuclei correlates with dystrophin expression in muscle fibers of immunosuppressed mdx host mice.

Feasibility, safety, and efficacy of myoblast transfer therapy on Duchenne muscular dystrophy boys.

Five billion normal myoblasts were injected into each of 21 Duchenne muscular dystrophy (DMD) boys aged 6-14 yr to assess the feasibility, safety, and efficacy of the Phase II myoblast transfer therapy (MTT). The Phase II study was designed to strengthen muscles of both lower limbs. Forty-eight intramuscular injections transferred the myoblasts into 22 major muscles at 55.6 x 10(6)/mL in 10 min under general anesthesia. Eleven boys had received 8 million myoblasts each 1 yr ago in the Phase I MTT. In the Phase II study, eight of them had their myoblasts subcultured from reserves frozen 1 yr ago. The donor myoblasts for each of the remaining boys were cultured from satellite cells derived from a 1-g muscle biopsy of a normal male who might or might not be histocompatible with the recipient. The immunosuppressant cyclosporine (Cy) is being administered to recipients for 6 mo after MTT to facilitate donor cell survival. There was no evidence of an adverse reaction to MTT or Cy as determined by serial laboratory evaluations including electrolytes, creatinine, and urea. Early objective functional tests using the KinCom Robotic Dynamometer were conducted on 13 subjects aged 6 to 13 before MTT and at 3 mo after MTT. Of the 69 muscle groups (knee extensors, knee flexors, plantar flexors) tested for isometric force generation in these subjects, 43% showed mean increase of 41.3% +/- 5.9 SEM, 38% showed no change, and 19% showed continuous force reduction of 23.4% +/- 3.1 SEM. The remaining subjects await the 3-mo post-MTT evaluation. The results indicate that 1) MTT is safe; 2) MTT increases muscle strength in DMD: 81% of the muscles tested showed either increase in strength or did not show continuous loss of strength; 3) more than 5 billion myoblasts can be cultured from 1 g normal muscle biopsy, providing unprecedented numbers of cells for MTT; 4) myoblasts, frozen over 1 yr, retain the ability to proliferate from 10 million to 5 billion, and to form normal myofibers; 5) injections of 5 billion myoblasts have not provoked any immunological rejection symptoms in the Phase II subjects, 11 of whom received 8 million myoblasts in the Phase I MTT a year ago; 6) it is safe to perform multiple injections of myoblasts into lower limb muscles without formation of emboli; and 7) donor cell rejection by the recipient can be prevented with Cy when properly managed.

Myoblast transfer therapy for Duchenne muscular dystrophy.

A randomly selected extensor digitorum brevis (EDB) muscle in each of three Duchenne muscular dystrophy (DMD) boys aged 9 to 10 was injected with approximately 8 x 10(6) myoblasts. The contralateral EDBs were sham-injected with carrier solution. Donor myoblasts were derived from cell culture of muscle biopsies from the normal ward or normal brothers of the recipients. Cyclosporine (CsA) treatment began two days before myoblast injection and continued for three months. Three days prior to myoblast injection and three months after, the isometric twitch and maximum voluntary contraction of the left and the right EDBs were measured. Myoblast-injected EDBs showed increases in tensions whereas sham-injected EDBs showed reductions. Both immunocytochemical staining and immunoblot revealed dystrophin in the myoblast-injected EDBs. Dystrophic characteristics such as fiber splitting, central nucleation, phagocytic necrosis, variation in fiber shape and size, and infiltration of fat and connective tissues were less frequently observed in these muscles. Sham-injected EDBs exhibited significant structural and functional degeneration and no dystrophin. Throughout the study, there was no sign of erythema, swelling or tenderness at the injection sites. Serial laboratory evaluation including electrolytes, creatinine, and urea did not reveal any significant changes before or after myoblast transfer. We conclude that myoblast transfer therapy is a safe and efficacious procedure to improve the biochemistry, structure, and function of degenerative EDB muscles in DMD.

Vital marker for muscle nuclei in myoblast transfer.

A new method is developed using Fluoro-Gold (FG) as a vital stain to label the nuclei of donor myoblasts in myoblast transfer studies. In vitro incubation with 0.01% FG for 16 h resulted in 100% nuclei labelling. Intensive fluorescence persisted following 9 days of subculture, when the human myoblasts were injected into the quadriceps of mouse recipients immunosuppressed with cyclosporine. Injected muscles showed mosaicism of host and donor nuclei 25 days after injection, indicating (i) survival and fusion among donor myoblasts, and (ii) fusion between host and donor cells. FG labelling was not observed in control muscles injected with an equal volume of FG-labelled dead myoblasts, 0.01% FG medium, or phosphate-buffered saline.