WEBVTT 00:00.000 --> 00:30.000 position:50% align:top line:0% *This machine-generated transcript may have errors. If remediation or a manually-generated transcript is needed, please contact NLM Support at https://support.nlm.nih.gov.* 00:08.369 --> 00:11.119 Dr Valerie New York University Medical 00:11.130 --> 00:11.659 Center, 00:11.670 --> 00:13.600 Human Muscle in tissue culture, 00:14.149 --> 00:16.940 neuromuscular diseases are considered to 00:16.950 --> 00:19.579 be disorders of the motor 00:19.590 --> 00:20.200 unit. 00:20.530 --> 00:22.750 It means the disease can 00:22.760 --> 00:25.190 originate either in 00:25.200 --> 00:28.110 spinal cord or in peripheral nerve 00:28.120 --> 00:30.219 or in the muscle fibers. 00:30.489 --> 00:33.310 With all techniques you for examination 00:33.319 --> 00:35.680 of muscle pathology like histochemistry, 00:35.689 --> 00:38.599 electron microscopy and electro biography. 00:38.759 --> 00:41.700 It is possible to identify involved in 00:41.709 --> 00:43.540 disease process system. 00:43.720 --> 00:44.470 However, 00:44.479 --> 00:47.419 pathogenesis of majority of neuromuscular 00:47.430 --> 00:50.330 diseases still remains to be known. 00:52.419 --> 00:55.130 Tissue culture of human muscle provides 00:55.139 --> 00:58.110 unique opportunity for examination 00:58.119 --> 01:01.099 of muscle which was developing in 01:01.110 --> 01:03.819 the environment where all other factors 01:03.830 --> 01:06.269 which possible are causing disease in 01:06.279 --> 01:08.389 humans are eliminated 01:09.220 --> 01:09.569 in, 01:09.580 --> 01:12.150 in the even in the muscle grown in such 01:12.160 --> 01:14.559 environment where neural humoral 01:14.569 --> 01:17.470 vascular and other factors are absolutely 01:17.480 --> 01:18.550 eliminated. 01:18.720 --> 01:21.669 We will be able to reproduce the changes 01:21.680 --> 01:23.010 present in the patient. 01:23.019 --> 01:24.110 Muscle biopsy. 01:24.400 --> 01:27.230 Primary character of muscle disorder 01:27.239 --> 01:28.790 could be anticipated. 01:30.660 --> 01:33.489 Multinucleate cross created 01:33.500 --> 01:35.879 muscle fiber in myogenesis 01:36.650 --> 01:39.330 in Vitra and in viva are the 01:39.339 --> 01:41.750 result of fusion of 01:42.050 --> 01:45.029 mononucleated spindle shaped myogenic 01:45.040 --> 01:45.690 cells. 01:45.930 --> 01:46.760 During myogenesis. 01:47.239 --> 01:49.839 Those cells attached to each other, 01:49.849 --> 01:51.730 they form multinucleate myotube 01:52.580 --> 01:55.269 and finally multi nucleated 01:55.279 --> 01:57.269 cross created muscle fiber 01:59.169 --> 02:01.760 growth of adult human muscle in 02:01.769 --> 02:04.599 Vitra is initiated by so called 02:04.610 --> 02:07.360 satellite cell described for the first 02:07.370 --> 02:10.320 time in 1961 by Alexander Mora from 02:10.330 --> 02:11.699 Rockefeller University. 02:12.259 --> 02:14.929 Satellite cells originate from 02:14.940 --> 02:17.470 embryonic myoblast during myogenesis. 02:18.039 --> 02:20.440 What happened is that all myoblast 02:20.449 --> 02:23.320 probably are originated from one pre 02:23.559 --> 02:24.190 myoblast. 02:24.199 --> 02:25.399 During myogenesis, 02:25.589 --> 02:26.770 they divide, 02:26.910 --> 02:28.589 they connect with each other, 02:28.600 --> 02:31.520 they fuse and they may cross trid muscle 02:31.529 --> 02:32.089 fiber. 02:32.389 --> 02:33.259 However, 02:33.270 --> 02:35.399 sometimes for any known reason, 02:35.520 --> 02:38.500 one single myoblast doesn't fuse and 02:38.509 --> 02:41.470 this cell gets incorporated under 02:41.479 --> 02:44.250 basement membrane of a dark muscle fiber 02:44.300 --> 02:46.800 and is forming so called satellite cell. 02:46.850 --> 02:49.449 Satellite cell is embryonic cell in the 02:49.460 --> 02:51.860 origin and is placed between 02:51.869 --> 02:54.360 basement and plasma membrane of dark 02:54.369 --> 02:54.979 muscle. 02:55.389 --> 02:57.839 Satellite cells account for about 02:57.850 --> 03:00.410 2% of all muscle nuclei 03:02.970 --> 03:05.589 even thought it was possible to 03:05.600 --> 03:08.589 grow embryonic chick muscle for many years. 03:08.899 --> 03:10.119 Only recently, 03:10.130 --> 03:12.979 it has become possible to grow adult 03:12.990 --> 03:13.979 human muscle. 03:14.279 --> 03:17.119 And two approaches for growing adult human 03:17.130 --> 03:18.839 muscle could be established. 03:19.059 --> 03:21.580 One is that whole muscle biopsy 03:21.589 --> 03:24.259 is sin and tissue culture is 03:24.270 --> 03:26.720 established from single cells. 03:26.960 --> 03:29.910 This technique appears to be very suitable 03:29.919 --> 03:32.309 for examining all steps of muscle 03:32.320 --> 03:33.250 development. 03:33.440 --> 03:34.130 Like here, 03:34.139 --> 03:36.990 we can see my house of single cells 03:37.000 --> 03:39.839 here they elongate and get attached to the 03:39.850 --> 03:41.389 bottom of the Petri dish. 03:41.460 --> 03:43.300 Here they divide again. 03:43.309 --> 03:44.250 And finally, 03:44.259 --> 03:46.880 this ammonia created spindle shaped 03:46.889 --> 03:49.160 cells are getting close together. 03:49.169 --> 03:50.160 In order to fuse, 03:52.190 --> 03:54.990 we can see here on higher magnification 03:55.000 --> 03:57.479 how two myoblast get together. 03:57.520 --> 03:59.220 There is a third myoblast, 03:59.229 --> 04:00.839 which was joining them. 04:00.850 --> 04:01.559 Finally, 04:01.570 --> 04:04.380 they make multinucleate myotube and 04:04.389 --> 04:06.199 cross created muscle fiber. 04:09.300 --> 04:12.229 Another approach which we prefer in 04:12.240 --> 04:14.889 our laboratory is to establish 04:14.899 --> 04:17.540 muscle culture from adult 04:17.549 --> 04:19.130 human muscle biopsy. 04:19.428 --> 04:21.678 We are establishing this making 04:21.688 --> 04:24.468 small one millimeter cubic ex 04:24.479 --> 04:26.678 plants which sometimes are cut 04:26.688 --> 04:29.558 longitudinally and sometimes are cut cross 04:29.859 --> 04:32.819 and we try to put about six or 04:32.829 --> 04:34.278 even at the present moment, 04:34.289 --> 04:36.959 more explant on the bottom of the 04:36.968 --> 04:39.479 gelatin and human plasma coated Petri 04:39.819 --> 04:40.218 dish. 04:43.119 --> 04:44.890 After a few days, 04:44.899 --> 04:47.119 about three or four days, 04:47.130 --> 04:49.880 we can observe that there are some single 04:49.890 --> 04:52.890 cells which are merging out from this original 04:52.899 --> 04:55.260 muscle X plan on higher power. 04:55.269 --> 04:58.079 One can observe that majority of the cells 04:58.089 --> 05:01.049 are Fibroplastic cells and 05:01.059 --> 05:03.839 very few are spindle long 05:03.850 --> 05:04.609 shaped, 05:04.619 --> 05:06.769 probably MYOB blastic cells. 05:07.079 --> 05:07.750 However, 05:07.760 --> 05:10.480 after about 10 days in in 05:10.600 --> 05:10.829 vitro, 05:11.000 --> 05:12.690 the cells fuse, 05:12.700 --> 05:14.869 they form multinucleate 05:15.619 --> 05:16.130 tubes, 05:16.459 --> 05:19.019 they mature become cross created. 05:19.029 --> 05:20.220 But unfortunately, 05:20.230 --> 05:21.730 after about three weeks, 05:22.019 --> 05:24.989 all these fibers are dying and they are become 05:25.000 --> 05:26.970 overgrown by connective tissue. 05:27.799 --> 05:30.640 This problem was bothering us a lot 05:30.649 --> 05:33.329 because our goal is to 05:33.339 --> 05:36.309 obtain very rich and mature 05:36.320 --> 05:37.929 muscle culture. 05:37.940 --> 05:40.329 In order to be able to apply all 05:40.339 --> 05:43.299 techniques used for examination of muscle 05:43.309 --> 05:44.010 biopsy, 05:44.019 --> 05:45.209 like histochemistry, 05:45.220 --> 05:48.010 electronic copy biochemistry and so on. 05:48.350 --> 05:49.359 For this reason, 05:49.369 --> 05:52.339 we were very pleased when we developed 05:52.350 --> 05:54.809 technique which we call plat 05:55.500 --> 05:57.420 explant re explantation technique, 05:57.489 --> 06:00.339 which enabled us to diminish 06:00.559 --> 06:03.309 amount of connective tissue in the cultural 06:03.320 --> 06:04.040 muscle. 06:05.309 --> 06:08.299 What we are doing is that we are picking up 06:08.320 --> 06:11.260 this original muscle explant to the 06:11.269 --> 06:13.529 entirely new Petri dish. 06:13.619 --> 06:16.380 And we are initiating like a new culture 06:16.390 --> 06:19.380 but from previously already present 06:19.390 --> 06:20.279 ex plants. 06:20.609 --> 06:23.019 And after about one or two days, 06:23.029 --> 06:25.690 single spindle shaped cells are 06:25.700 --> 06:28.350 coming out from this plat which 06:28.359 --> 06:30.959 fuses and after a few days they form multi 06:30.970 --> 06:32.640 nucleated mao tubes. 06:32.920 --> 06:35.420 And here we can see like amount of 06:35.429 --> 06:38.329 connective tissue was greatly reduced in the sculpture. 06:38.339 --> 06:40.000 All this staining is the P N H. 06:40.010 --> 06:42.720 The heterogenous staining are presenting 06:42.730 --> 06:45.079 multi nucleated muscle fibers. 06:45.089 --> 06:47.869 And this picture was taking about after eight 06:47.880 --> 06:49.160 weeks in Vitra 06:52.339 --> 06:54.959 using explant explantation technique. 06:54.970 --> 06:57.450 We are achieving quite advanced maturation. 06:57.459 --> 07:00.010 We can see that muscle fibers are 07:00.019 --> 07:02.829 becoming cross created and majority of the new 07:02.839 --> 07:05.410 climate migrate to the periphery of the fiber. 07:08.869 --> 07:10.369 Like I said before, 07:10.380 --> 07:13.019 we are attempting to 07:13.029 --> 07:15.929 apply all techniques using for examination 07:15.940 --> 07:18.510 of muscle biopsy to the culture human 07:18.519 --> 07:19.100 muscle. 07:19.529 --> 07:21.950 And uh I would like to show a few 07:21.959 --> 07:24.290 examples how we are doing this. 07:24.299 --> 07:27.049 One approach is to air dry, 07:27.059 --> 07:29.679 entire culture including original 07:29.690 --> 07:32.279 explant and apply histo chemical 07:32.290 --> 07:32.769 reaction. 07:32.779 --> 07:33.769 On this example, 07:33.779 --> 07:36.325 the P N H we can see on 07:36.334 --> 07:38.125 lower low power, 07:38.135 --> 07:40.894 many many fibers coming out from the egg plant. 07:40.904 --> 07:43.815 And here longitudinal view on higher power, 07:43.825 --> 07:46.815 very strong staining of cultural human muscle, 07:46.825 --> 07:49.394 only nuclear area remains in stain. 07:49.795 --> 07:52.325 Another approach is to pick up 07:52.334 --> 07:55.029 entire outgrowth and 07:55.040 --> 07:57.320 leaving original X men alone. 07:57.559 --> 08:00.260 And put this outgrowths cross on the top 08:00.350 --> 08:02.390 of the adult muscle fiber, 08:02.470 --> 08:05.000 we freeze this and we perform serial cross 08:05.010 --> 08:05.760 sections. 08:08.149 --> 08:11.130 This light illustrates like cult 08:11.220 --> 08:13.880 human muscle are very strongly positively 08:13.890 --> 08:15.700 stained with A T P S reaction. 08:15.709 --> 08:18.480 Perform at P H 9.4 A 08:18.489 --> 08:21.230 T P A reaction perform after acid 08:21.239 --> 08:22.239 preincubation. 08:22.420 --> 08:25.160 Suing the heroes give somehow 08:25.359 --> 08:28.040 weaker staining and phosphor staining 08:28.049 --> 08:30.760 appears very characteristic in farmers of 08:30.769 --> 08:33.440 spots or legs which we like to call them. 08:33.450 --> 08:35.890 Those legs are positive stains or phosphor. 08:38.479 --> 08:40.948 As we all know that 08:41.158 --> 08:44.049 human and animal muscle is differentiated for 08:44.059 --> 08:46.348 different histo chemical fiber types. 08:46.528 --> 08:49.468 We can see on this example when A T P S reaction 08:49.479 --> 08:52.388 at P H 9.4 is performed that this 08:52.398 --> 08:55.208 muscle majority on this muscle are staying dark 08:55.218 --> 08:57.559 indicating that there are type two fibers 08:57.588 --> 09:00.588 reverse being through with A P A reaction 09:00.598 --> 09:02.989 performed after acid preincubation. 09:03.309 --> 09:04.239 However, 09:04.250 --> 09:06.849 those two light fibers here are staying 09:06.859 --> 09:09.280 dark here indicating that they belong to 09:09.289 --> 09:11.820 different uh group of fiber types. 09:12.000 --> 09:13.280 And here with D P N H, 09:13.289 --> 09:13.969 the heterogenous, 09:14.020 --> 09:16.289 we are able to identify types 09:16.299 --> 09:18.299 of uh those two groups. 09:18.619 --> 09:19.500 However, 09:19.510 --> 09:22.289 when car master were put on the 09:22.299 --> 09:25.080 top of the sedar master and serial cross 09:25.090 --> 09:27.429 sections were performed, 09:27.440 --> 09:30.059 we were not able to observe 09:30.070 --> 09:32.549 reciprocal reaction between 09:32.559 --> 09:34.479 different histo chemical staining. 09:35.049 --> 09:37.549 And this fact indicates that 09:37.559 --> 09:40.210 cultural muscle grown in 09:40.320 --> 09:42.739 Vitra without neural influence, 09:42.750 --> 09:45.059 are not able to differentiate for his 09:45.070 --> 09:47.630 different histo chemical fiber 09:47.640 --> 09:48.390 types. 09:51.380 --> 09:54.190 For electro microscopical examination, 09:54.200 --> 09:57.190 we like to fix entire 09:57.200 --> 10:00.020 dish with muscle cultures inside 10:00.030 --> 10:02.409 with grower aldehyde and post in. 10:03.150 --> 10:05.710 And then we embedded we are embedding in. 10:06.750 --> 10:09.559 We found extremely necessary not 10:09.570 --> 10:12.239 to perform blind studies on 10:12.250 --> 10:13.309 the the culture, 10:13.979 --> 10:16.179 but to be able to perform selective 10:16.190 --> 10:18.890 examination of culture for electron microscopical 10:18.900 --> 10:19.830 examination. 10:20.099 --> 10:23.000 What we are like to do is after we 10:23.739 --> 10:24.950 embed the dish, 10:24.960 --> 10:26.479 we stain this with metal and blow, 10:26.840 --> 10:29.239 We observe fibers of our 10:29.250 --> 10:30.210 interest. 10:30.219 --> 10:32.909 We mark them under the bottom of the dish. 10:33.200 --> 10:35.929 And then using by developed by us 10:35.940 --> 10:38.479 technique which we call drilling technique, 10:38.640 --> 10:41.489 we select against this area and we pass them 10:41.500 --> 10:42.080 out. 10:42.260 --> 10:45.250 And this small disk comes from this big 10:45.260 --> 10:48.179 disk here and shows the same fibers which were pre 10:48.239 --> 10:50.830 previously selected and shown here. 10:51.859 --> 10:54.609 We put this whole small disk on the track, 10:54.619 --> 10:55.409 we trim this, 10:55.419 --> 10:58.330 we cut the thin section and e examine under electronics. 10:58.880 --> 11:01.390 It is extremely important 11:01.400 --> 11:03.580 perform to perform 11:03.609 --> 11:06.489 selective examination of fibers for electronics 11:07.510 --> 11:09.989 because as we all know in the disease 11:10.000 --> 11:10.710 process, 11:10.719 --> 11:11.630 very often, 11:11.640 --> 11:14.559 not entire fiber is affected by the 11:14.570 --> 11:17.270 disease process and very often in one 11:17.280 --> 11:18.570 entire muscle biopsy, 11:18.580 --> 11:20.840 only very few fibers are affected. 11:20.880 --> 11:23.609 And the same is true in the tissue culture 11:23.619 --> 11:24.239 fibers. 11:24.330 --> 11:27.330 This is why we found this extremely important to 11:27.340 --> 11:30.250 be able to selectively examine cultural muscle. 11:30.260 --> 11:31.799 For electron microscopy. 11:34.890 --> 11:36.539 Using this drilling technique, 11:36.549 --> 11:39.500 we are able to pick nuclei area. 11:39.510 --> 11:42.349 If we are specifically interested in this or we are 11:42.359 --> 11:45.309 able to decide that we would like to look at 11:45.320 --> 11:46.950 the periphery of the fiber. 11:47.020 --> 11:48.530 And we are really, 11:48.539 --> 11:50.880 but after small in section are cut, 11:50.890 --> 11:53.890 we really know where we are and what structures we 11:53.900 --> 11:54.559 examine. 11:55.070 --> 11:57.869 And this picture here shows very young multi 11:58.099 --> 12:01.039 nucleated area of very young my tubes 12:01.070 --> 12:03.799 with some beginning of myofibril formation 12:03.809 --> 12:06.109 here on the periphery from two sides. 12:06.390 --> 12:07.059 However, 12:07.070 --> 12:09.510 this fiber is much more mature. 12:09.520 --> 12:12.500 We can see much better organization of myo 12:12.719 --> 12:15.559 fibras here and we can 12:15.570 --> 12:18.130 see beginning of zen formation here is not 12:18.140 --> 12:20.489 even nice zen 12:20.760 --> 12:23.419 present the same on this side and 12:23.429 --> 12:26.210 mitochondria are present and rough in the plasm cri. 12:27.070 --> 12:27.900 However, 12:27.909 --> 12:30.419 we can see that even this fiber is very young 12:30.429 --> 12:33.390 because center of this fiber is loaded by 12:33.400 --> 12:34.830 ribosome rough in the plasm cri. 12:35.489 --> 12:38.140 And all this um signs 12:38.150 --> 12:40.260 indicate that it is very young, 12:40.270 --> 12:42.559 actively developing muscle fiber. 12:45.039 --> 12:45.969 However, 12:45.979 --> 12:48.780 this picture shows us much 12:48.789 --> 12:51.539 more mature about six weeks in Vitra 12:51.780 --> 12:54.500 mass fiber which is able to contract and 12:54.510 --> 12:57.130 maintain in this form for a longer period of time. 12:57.140 --> 12:59.830 You can see nicely organized my filaments and Zs 13:00.239 --> 13:01.890 formation is present. 13:04.820 --> 13:07.080 As we all know Aceon 13:07.090 --> 13:09.830 receptors in normal muscle 13:09.989 --> 13:12.250 are localized to post synaptic 13:12.260 --> 13:13.210 membrane. 13:13.549 --> 13:16.070 In endplate area of the muscle, 13:16.289 --> 13:19.210 we can never see any aceto colon receptor 13:19.219 --> 13:20.260 extra junction. 13:20.559 --> 13:23.330 They are all located in the end plate 13:23.340 --> 13:24.070 area. 13:24.880 --> 13:27.250 Visualization of alaia 13:27.260 --> 13:29.640 receptors became much easier 13:29.659 --> 13:32.630 after immunoperoxidase staining 13:32.640 --> 13:35.130 of alpha Banga toxin was 13:35.140 --> 13:37.869 performed first by Daniels and Vogel 13:38.059 --> 13:40.270 and it became later used. 13:40.549 --> 13:42.260 What this staining um 13:42.270 --> 13:45.059 enables us to see is 13:45.070 --> 13:47.729 immunology reaction after 13:47.739 --> 13:50.630 alpha Bangar toxin obtained from the venom 13:50.640 --> 13:51.960 of snake multi 13:52.820 --> 13:55.590 binds to a receptors 13:55.890 --> 13:58.479 and then antibodies against alpha Bangar 13:58.659 --> 14:00.390 toxin are applied. 14:00.409 --> 14:02.929 They combine and immuno peroxidase 14:02.940 --> 14:05.780 staining is applied to the end to visualize this 14:05.789 --> 14:06.450 reaction. 14:06.809 --> 14:09.549 And we can see here and plate of the 14:09.559 --> 14:11.830 normal master showing that 14:11.840 --> 14:14.580 postsynaptic membrane is darkly stain. 14:14.840 --> 14:17.750 And there is present staining 14:17.760 --> 14:20.130 of presynaptic area as well 14:21.489 --> 14:22.599 as a normal muscle. 14:22.609 --> 14:24.619 Like I mentioned previously, 14:24.630 --> 14:27.159 only end plate area gives 14:27.169 --> 14:28.409 positive staining. 14:28.609 --> 14:29.469 However, 14:29.479 --> 14:31.799 when muscle is denervated, 14:32.099 --> 14:34.979 like we can see here there is the few 14:34.989 --> 14:37.130 staining extra junction. 14:37.500 --> 14:40.309 So we can see that there are a receptors 14:40.320 --> 14:43.140 not only present in the end plate area, 14:43.179 --> 14:45.760 but they are present all over the membrane 14:46.609 --> 14:49.419 is a difference to normal master here where 14:49.429 --> 14:51.989 extra junctional area remains in 14:52.000 --> 14:52.539 stain. 14:52.849 --> 14:55.630 This work was done uh with cooperation of 14:55.640 --> 14:58.530 doctor um bender and the 14:58.659 --> 14:58.719 ringer, 15:00.010 --> 15:01.729 we were interested to see 15:02.559 --> 15:05.070 if a neurally grown 15:05.380 --> 15:08.030 culture muscle will have 15:08.039 --> 15:09.900 similar appearance of 15:10.609 --> 15:12.599 receptors to 15:12.929 --> 15:15.369 biopsy muscle or it will have 15:15.380 --> 15:18.190 similar appearance of receptors to 15:18.200 --> 15:20.989 denervated muscle or it will be entirely 15:21.000 --> 15:21.979 different story. 15:22.250 --> 15:25.140 And what we have done we applied 15:25.150 --> 15:28.010 immuno perox staining to a 15:28.020 --> 15:29.960 neurally grown culture. 15:29.969 --> 15:32.880 And this picture represent rat 15:32.890 --> 15:33.989 culture muscle. 15:34.000 --> 15:35.320 And you can see here, 15:35.530 --> 15:37.239 diffuse staining 15:38.250 --> 15:41.020 Oxin indicating a receptor 15:41.030 --> 15:43.650 binding site on this culture, 15:43.659 --> 15:44.679 muscle fiber. 15:47.299 --> 15:49.650 Those two slides shows us 15:49.659 --> 15:52.460 show us staining of cultural 15:52.469 --> 15:55.059 human muscle here and 15:55.070 --> 15:57.799 here this immuno peroxidase 15:57.960 --> 16:00.150 staining of ace receptors, 16:00.440 --> 16:01.309 the insides. 16:03.340 --> 16:06.070 And this is electron microscopical picture 16:06.429 --> 16:09.349 which shows diffuse 16:09.359 --> 16:12.109 staining of the plasma lemma. 16:13.150 --> 16:15.799 Those are two adjacent fibers 16:17.000 --> 16:18.799 of human culture. 16:19.140 --> 16:21.979 Whole preparation is and only I 16:23.200 --> 16:23.820 is performed. 16:25.840 --> 16:28.450 And this slide shows comparison 16:28.460 --> 16:31.349 between immuno perox of 16:31.419 --> 16:33.830 alpha toxin in the human culture, 16:33.840 --> 16:34.450 muscle, 16:34.739 --> 16:35.799 in the rat culture, 16:35.809 --> 16:38.669 muscle and in the chicken culture muscle. 16:39.969 --> 16:42.510 So as we can see when 16:42.969 --> 16:45.349 muscles are growing without nerve 16:45.359 --> 16:46.500 influence, 16:46.520 --> 16:49.320 they do not differentiate for different histo chemical 16:49.330 --> 16:50.299 fiber types. 16:50.489 --> 16:52.859 And as with respect to a 16:53.469 --> 16:54.419 receptors, 16:54.479 --> 16:57.419 they behave like denervated muscle 16:57.429 --> 17:00.169 in viva having Aceon receptor 17:00.179 --> 17:02.299 spread in the entire area of 17:02.950 --> 17:03.929 tic membrane. 17:05.659 --> 17:08.609 We are interested to see if 17:08.619 --> 17:11.509 muscle in culture will be innervated if 17:11.519 --> 17:14.519 there will be localized area of Aceon 17:14.529 --> 17:15.479 receptors. 17:15.548 --> 17:18.468 And what we do now this preparation shows 17:18.479 --> 17:21.458 is this is human muscle in tissue culture, 17:21.609 --> 17:24.249 which we are trying to innervate 17:24.259 --> 17:26.769 by embryonic rat spinal 17:26.779 --> 17:29.099 cord here are nerve 17:29.109 --> 17:31.310 coming to this human muscle. 17:31.319 --> 17:34.109 These studies are in progress now and 17:34.119 --> 17:36.989 we are going to search 17:37.000 --> 17:39.439 for endplate formation in this 17:39.449 --> 17:40.119 culture. 17:40.130 --> 17:40.670 Muscle. 17:43.969 --> 17:44.790 In general, 17:44.800 --> 17:47.209 one can see following 17:47.219 --> 17:49.439 goals in culturing human muscle 17:49.449 --> 17:50.270 fibers. 17:51.839 --> 17:54.839 We one would like to see in abnormal 17:54.849 --> 17:57.829 muscle if it would be possible 17:57.939 --> 18:00.750 to reincarnate a characteristic 18:00.760 --> 18:03.709 morphological defect which is present in 18:03.719 --> 18:06.339 muscle biopsy to the culture muscle. 18:06.880 --> 18:09.239 Another thing it would be possible to 18:09.250 --> 18:12.250 reincarnate a characteristic biochemical 18:12.260 --> 18:15.209 defect which is present in the muscle biopsy into the 18:15.219 --> 18:16.109 cultural muscle. 18:16.949 --> 18:18.089 And third point, 18:18.099 --> 18:20.709 very important is if it would be possible to 18:20.719 --> 18:23.619 prevent or to reverse the defect by 18:23.630 --> 18:26.300 in Vitra treatment in normal 18:26.310 --> 18:26.979 muscle. 18:27.219 --> 18:30.199 I started kind of like from the end before and 18:30.209 --> 18:32.839 I analyzed the normal appearance of muscle 18:32.849 --> 18:35.300 fiber and then we can discuss later 18:35.310 --> 18:38.310 inducing of characteristic defect in normal muscle 18:38.319 --> 18:38.900 fibers. 18:39.290 --> 18:42.199 I would like to start with some 18:42.209 --> 18:45.140 of our positive data when we were able 18:45.150 --> 18:48.069 to reproduce some 18:48.079 --> 18:51.030 morphologic defect present in muscle 18:51.040 --> 18:52.390 biopsy into the culture, 18:52.400 --> 18:52.880 muscle. 18:56.160 --> 18:58.800 We started to grow muscle from eight 18:58.810 --> 19:00.510 patients with ophthalmaplegia 19:01.219 --> 19:02.260 syndrome. 19:02.270 --> 19:05.189 Ophthalmoplegic syndrome is characterized by all 19:05.199 --> 19:08.089 this uh data 19:08.099 --> 19:10.660 which are put here on this slide and 19:10.670 --> 19:13.150 morphologically they are characterized by 19:13.160 --> 19:16.060 ragged red fibers present in the muscle 19:16.069 --> 19:16.770 biopsy. 19:19.270 --> 19:21.640 Ragged red fibers are the best 19:21.650 --> 19:24.140 visualized by trichrome modified Trichrome 19:24.209 --> 19:27.130 staining which shows red ring around the 19:27.140 --> 19:29.640 fiber and by succeeding the heterogenous 19:29.650 --> 19:32.349 staining which is showing very dark rink 19:32.359 --> 19:34.770 around the fiber on the electro 19:34.800 --> 19:36.650 microscopical examination. 19:36.979 --> 19:39.709 One can see that they are big 19:39.719 --> 19:42.069 collection of abnormal 19:42.079 --> 19:43.069 mitochondria. 19:43.209 --> 19:45.099 They are very peculiar pattern. 19:45.109 --> 19:46.290 They are enlarged, 19:46.300 --> 19:47.469 they are in cluster, 19:47.800 --> 19:49.550 some have crested, 19:49.560 --> 19:51.949 twisted in fingerprint pattern, 19:52.119 --> 19:55.030 some are filled with amorphous material here 19:55.040 --> 19:56.349 and here and here. 19:56.359 --> 19:59.069 Some have para crystal included in it 19:59.250 --> 19:59.949 in general. 19:59.959 --> 20:02.930 They are enlarged in a mouth and in size 20:02.939 --> 20:04.459 and they are put in clusters. 20:06.670 --> 20:09.439 Cyto oxidase is 20:09.449 --> 20:12.250 very useful in examination of 20:12.260 --> 20:15.089 muscle mitochondria because we don't 20:15.099 --> 20:17.719 have to pay attention to the entire tissue. 20:17.729 --> 20:20.569 And only mitochondria are stain cytochrome 20:20.780 --> 20:23.780 oxidase stain out the membrane of 20:23.790 --> 20:26.020 mitochondria and cry. 20:26.689 --> 20:28.439 This is normal human muscle 20:30.839 --> 20:33.579 and this light demonstrate muscle 20:33.589 --> 20:36.319 biopsy from patients with ragged fiber and cyto 20:36.810 --> 20:38.469 oxidase staining form. 20:38.719 --> 20:41.339 We can see abnormal mitochondria 20:41.349 --> 20:41.869 here, 20:41.880 --> 20:44.709 crystal crystal not being stained with cyto 20:45.170 --> 20:47.979 oxidase but outer membrane 20:47.989 --> 20:50.780 and reminder structures are 20:50.790 --> 20:51.369 stain 20:54.699 --> 20:57.420 when tissue cultures were established from 20:57.430 --> 20:58.209 muscle biopsy. 20:58.219 --> 21:00.209 From patients with ragged red fibers. 21:00.219 --> 21:02.810 We haven't noticed any difference in the 21:02.819 --> 21:05.430 ratio of growth time of forming of 21:05.439 --> 21:07.849 myotube or maturation of muscle fiber. 21:08.060 --> 21:10.579 We can see here very rich outgrowth from the 21:10.589 --> 21:12.270 original muscle biopsy, 21:12.420 --> 21:14.989 we can see very nice developed cross 21:15.380 --> 21:17.750 trid mature master fibers. 21:18.030 --> 21:19.040 However, 21:19.050 --> 21:20.520 in some of the fibers, 21:20.530 --> 21:22.680 there were some collection of gran 21:22.709 --> 21:25.699 material present which we were 21:25.709 --> 21:28.569 not able of course to identify on 21:28.579 --> 21:30.449 the light microscopical level. 21:30.800 --> 21:31.569 However, 21:31.579 --> 21:34.219 when cytochrome oxidase reaction was 21:34.229 --> 21:36.069 performed on the sculptural muscle, 21:36.290 --> 21:38.839 we kind of noticed that collection 21:39.050 --> 21:41.650 of this granu materials stay 21:41.660 --> 21:43.699 very richly with cyto oxidase 21:44.130 --> 21:46.989 indicating that they might be mitochondrial 21:47.020 --> 21:47.910 in the original 21:50.500 --> 21:52.989 fact that only very few 21:53.000 --> 21:55.479 fibers are stained are 21:55.619 --> 21:58.439 abnormal in this muscle ground 21:58.449 --> 22:00.959 from patients with rare fibers is not really 22:00.969 --> 22:03.810 surprising because when we look in 22:03.819 --> 22:05.430 this three example, 22:05.439 --> 22:07.989 from different patients with rare fibers, 22:08.339 --> 22:09.869 we can see that the 22:10.469 --> 22:13.469 fibers are really very few 22:14.930 --> 22:17.239 and the entire muscle looks 22:17.250 --> 22:18.979 quite normal. 22:19.199 --> 22:22.079 So when we expect pathological changes 22:22.089 --> 22:24.920 coming only from this very few fibers, 22:24.930 --> 22:27.469 we really wouldn't expect entire dish of 22:27.479 --> 22:29.099 culture to be affected 22:32.449 --> 22:34.689 when using this breeding technique, 22:34.699 --> 22:37.500 which I explained before we selected 22:37.510 --> 22:40.489 area of this rich cyto oxidase staining. 22:40.880 --> 22:43.859 And the we examine this 22:43.869 --> 22:45.780 area under electron microscopy, 22:45.859 --> 22:48.530 we indeed observed that this area 22:48.540 --> 22:51.069 corresponds to abnormal looking mitochondria. 22:51.469 --> 22:54.189 And here we can see from cultural master giant 22:54.199 --> 22:56.660 mitochondrial filled with amorphous 22:56.670 --> 22:59.140 material and very peculiar shape, 22:59.150 --> 23:00.250 peculiarly shaped. 23:00.530 --> 23:03.520 And there are some crystal stain positively 23:03.530 --> 23:05.979 with cyto oxidase on the edge of this 23:05.989 --> 23:08.920 fiber of this mitochondrial here and here 23:08.930 --> 23:11.400 too and kind of like amorphous 23:11.410 --> 23:13.060 material and stain 23:14.630 --> 23:17.140 is being in the center as 23:17.150 --> 23:19.900 compared to normal cultural mitochondria 23:21.459 --> 23:23.780 which are small and very rich in Christian. 23:25.400 --> 23:27.859 This next four slides 23:28.180 --> 23:30.869 will show similarities which we often 23:30.880 --> 23:33.359 observe between mitochondria 23:33.369 --> 23:36.170 present in the given patient muscle biopsy 23:36.250 --> 23:38.900 and in the culture grown from this 23:38.910 --> 23:39.890 patient muscle. 23:40.319 --> 23:42.530 And uh if you could just look at this 23:42.540 --> 23:43.890 mitochondrial here, 23:43.900 --> 23:46.209 we can see very peculiar shape 23:46.300 --> 23:49.000 part is filled with amorphous material. 23:49.199 --> 23:52.150 There are some crest that twisted in peculiar 23:52.160 --> 23:55.099 pattern and there is like another small mitochondrial in 23:55.109 --> 23:56.459 the center of this big one. 23:56.680 --> 23:58.589 This is patient muscle biopsy. 24:00.790 --> 24:03.410 This is this patient muscle 24:03.420 --> 24:04.829 grown in culture. 24:04.839 --> 24:07.699 And we can see very similar mitochondria 24:07.709 --> 24:10.290 here to this one which we observed 24:10.300 --> 24:13.040 before and in the neighborhood of this 24:13.050 --> 24:14.829 abnormal looking mitochondria. 24:14.890 --> 24:17.760 There are some mitochondria which are quite normal in 24:17.770 --> 24:18.579 appearance. 24:20.239 --> 24:22.579 And here is another example beside 24:23.400 --> 24:24.780 stain mitochondria. 24:24.790 --> 24:26.780 Here from patient master biopsy, 24:26.790 --> 24:29.780 we can see cry being packed here in the 24:29.790 --> 24:32.140 center and here being packed in the 24:32.150 --> 24:35.020 periphery in very peculiar fashion. 24:35.430 --> 24:38.010 And from this patient culture master, 24:38.400 --> 24:40.949 we can see that mitochondria appear in 24:40.959 --> 24:42.489 very similar pattern, 24:42.500 --> 24:43.550 cry packed, 24:43.560 --> 24:46.109 strongly stained and center 24:46.400 --> 24:49.290 being and stain and filled with amorphous material. 24:51.329 --> 24:52.380 However, 24:52.390 --> 24:55.189 we will never be able reproduced 24:55.359 --> 24:58.000 as far para 24:58.010 --> 25:00.520 crystal inclusions which are 25:00.530 --> 25:03.430 always almost present in the abnormal 25:03.439 --> 25:05.449 mitochondria and red fibers. 25:05.630 --> 25:08.479 Here is like exaggerated example of big 25:08.489 --> 25:11.219 paracin inclusion present in patient 25:11.229 --> 25:12.260 master biopsy. 25:12.270 --> 25:14.699 We we were able to reproduce 25:15.130 --> 25:17.930 abnormal mitochondria as far to the 25:17.939 --> 25:19.729 size and shape is concerned, 25:19.739 --> 25:22.609 but we we're not able to reproduce parasol 25:22.979 --> 25:25.060 and inclusion probably 25:25.469 --> 25:28.290 much longer time is necessary for parasol 25:28.680 --> 25:31.500 inclusion to develop than we are dealing 25:31.640 --> 25:33.310 in tissue culture condition. 25:37.050 --> 25:39.969 Another step forward which I would 25:39.979 --> 25:42.140 like to discuss now is 25:42.150 --> 25:45.130 our ability to in one 25:45.140 --> 25:47.660 case to reincarnate in culture 25:48.540 --> 25:51.390 characteristic morphological defect 25:51.400 --> 25:54.160 and partially biochemical defect 25:54.170 --> 25:56.750 which was present in patient muscle biopsy. 25:59.640 --> 26:02.099 This man was very 26:02.109 --> 26:04.660 healthy until age 22 26:05.160 --> 26:06.530 he was in the Marine Corps. 26:07.189 --> 26:08.130 However, 26:08.140 --> 26:10.569 at age 22 he started to feel 26:10.579 --> 26:13.079 weak and now he has from 12 26:13.089 --> 26:13.770 years, 26:13.780 --> 26:16.780 slowly progressive proximal muscle 26:16.790 --> 26:17.530 weakness, 26:17.540 --> 26:20.410 but some distal muscles are involved as well. 26:20.420 --> 26:22.890 Otherwise he doesn't have any complaints 26:24.680 --> 26:26.599 in muscle biopsy of this patient. 26:26.609 --> 26:29.560 We could observe vacation present 26:29.569 --> 26:30.709 in some fibers, 26:32.930 --> 26:34.449 electron microscopically, 26:34.459 --> 26:37.349 his muscle was badly damaged and 26:37.359 --> 26:40.140 we can see it was very peculiar fashion. 26:40.819 --> 26:41.430 It was in. 26:41.439 --> 26:43.810 There were inclusion of 26:44.250 --> 26:45.900 multi laminated 26:46.800 --> 26:48.770 bodies here and here. 26:48.780 --> 26:50.130 Some lightly stain, 26:50.140 --> 26:52.609 some darkly stain but on cross 26:52.619 --> 26:55.290 section remained and look 26:55.300 --> 26:57.650 like cross section of the cabbage. 26:58.010 --> 26:59.709 This master was hardly destroyed, 26:59.719 --> 27:02.569 very destroyed and we couldn't observe 27:02.880 --> 27:05.250 an accumulation of glycogen there. 27:07.359 --> 27:09.849 When tissue cultures were established from this 27:09.859 --> 27:12.119 patient muscle biopsy, 27:12.400 --> 27:15.250 we could notice that even very 27:15.260 --> 27:17.939 single and young cells already were highly 27:17.949 --> 27:20.650 vacated and this vacation were 27:20.660 --> 27:23.390 carry on till muscle was quite 27:23.400 --> 27:23.930 mature. 27:23.939 --> 27:25.119 And we can see here, 27:25.130 --> 27:28.119 multinucleate mature myotube, 27:28.130 --> 27:31.060 highly evacuated stain preparation and 27:31.069 --> 27:31.699 end stain. 27:31.709 --> 27:33.459 All these vacuums present 27:35.920 --> 27:37.689 electron microscopically. 27:37.699 --> 27:40.699 The sculpture fiber similarly to 27:40.709 --> 27:43.270 his muscle biopsy was very 27:43.280 --> 27:43.920 damaged. 27:43.930 --> 27:46.640 And we could see all this field is 27:46.650 --> 27:49.420 loaded by these vacuums which under electron 27:49.430 --> 27:52.209 microscopy appear like multi laminated 27:52.719 --> 27:53.689 bodies, 27:54.250 --> 27:57.050 bitter mind cross section of the cabbage 27:57.060 --> 27:57.810 as well. 27:58.140 --> 28:00.770 This muscle culture was so damaged that 28:00.780 --> 28:03.560 I couldn't really find any muscle which 28:03.569 --> 28:04.780 was normal looking. 28:04.790 --> 28:07.469 Every muscle fiber which I choose for electronics 28:08.119 --> 28:10.760 examination was loaded by this 28:10.770 --> 28:11.829 abnormalities. 28:12.449 --> 28:13.430 Biochemically. 28:13.439 --> 28:16.030 Doctor Salvador De from 28:16.040 --> 28:18.270 Columbia University Medical Center, 28:19.290 --> 28:21.920 examine muscle biopsy of this patient 28:21.930 --> 28:23.619 and his cultural muscle. 28:23.630 --> 28:26.339 And he found in both cases highly 28:26.349 --> 28:27.670 elevated acid phosphatase. 28:28.310 --> 28:28.959 However, 28:28.969 --> 28:31.780 specific biochemical defect probably 28:31.790 --> 28:34.689 lies which is responsible for these 28:34.699 --> 28:37.689 changes in both this patient cultural muscle and in his 28:37.699 --> 28:40.260 muscle biopsy still remains unknown. 28:42.130 --> 28:43.760 As the next example, 28:43.880 --> 28:46.140 I would like to show 28:47.319 --> 28:49.339 it two 28:49.719 --> 28:52.349 different forms of glycogen 28:52.430 --> 28:53.890 storage diseases. 28:54.250 --> 28:56.599 In one form when we were able to 28:56.729 --> 28:58.979 reincarnate in culture, 28:59.170 --> 29:02.089 characteristic morphologic and biochemical 29:02.099 --> 29:04.910 defect present in the patient's muscle biopsy. 29:05.239 --> 29:08.189 And one example of um glycogen 29:08.280 --> 29:09.319 storage disease, 29:09.329 --> 29:12.010 which we were not able to reincarnate um 29:12.020 --> 29:13.380 defect 29:16.369 --> 29:17.380 as we know 29:19.089 --> 29:22.060 breaks the glucose through two 29:22.180 --> 29:22.969 pathway. 29:22.979 --> 29:25.719 Major pathways is through phosphorylase. 29:25.979 --> 29:28.479 And minor pathway is through alpha 29:28.489 --> 29:31.140 glucosidase called acid as 29:31.150 --> 29:31.579 well. 29:31.849 --> 29:34.800 So alpha glucosidase works here and 29:34.810 --> 29:36.770 breaks glycogen to glucose. 29:36.780 --> 29:39.560 And phosphorylase works as a major 29:39.569 --> 29:42.270 pathway in macarthur 29:42.410 --> 29:43.420 disease. 29:43.939 --> 29:46.839 Myoor is absent in the 29:46.849 --> 29:49.680 patient muscle and this slide shows 29:49.689 --> 29:52.369 negative staining of phosphorylase 29:52.670 --> 29:54.160 from muscle biopsy, 29:54.170 --> 29:56.869 from patient with me disease, 29:56.880 --> 29:58.010 so called myoor 29:58.750 --> 29:59.920 deficiency. 30:00.650 --> 30:03.280 Because phosphorylase is absent from this 30:03.290 --> 30:06.040 muscle glycogen cannot break to 30:06.050 --> 30:08.979 glucose in this muscle and is accumulating 30:09.170 --> 30:11.959 hardly as a cytoplasmic 30:11.969 --> 30:12.369 glycogen. 30:12.500 --> 30:13.819 In this patient, 30:13.829 --> 30:14.630 muscle biopsy, 30:14.640 --> 30:17.319 what could be visualized under electron microscopy. 30:18.790 --> 30:20.000 However, 30:20.349 --> 30:23.219 interesting enough when we were growing 30:23.670 --> 30:26.520 muscle from patient with mac 30:26.770 --> 30:27.599 disease, 30:27.680 --> 30:30.260 what was previously reported by uh 30:30.270 --> 30:33.079 doctor doctor and as 30:33.089 --> 30:33.520 well, 30:34.130 --> 30:36.180 we were able to detect phosphorylase 30:36.689 --> 30:39.260 staining histo chemically here is 30:39.270 --> 30:40.959 rich rich outgrowth. 30:41.790 --> 30:44.479 All these fibers are cultural fiber from patient 30:44.489 --> 30:46.900 with um phosphor deficiency. 30:47.189 --> 30:48.609 And we can see here, 30:48.619 --> 30:51.609 typical characteristic of a standing 30:51.619 --> 30:52.719 for master culture. 30:53.680 --> 30:56.599 We made like further 30:56.609 --> 30:59.369 examination in this area and 30:59.380 --> 31:01.949 we have done electro microscopical 31:01.959 --> 31:04.800 studies of culture muscle from patients with mac 31:04.979 --> 31:05.750 disease. 31:06.140 --> 31:09.089 And there we couldn't find any 31:09.324 --> 31:10.255 normal is either, 31:10.265 --> 31:12.665 we haven't seen any accumulation of glycogen 31:13.204 --> 31:15.935 mas was becoming cross rated and very 31:15.944 --> 31:17.084 nicely developed. 31:17.324 --> 31:18.084 And again, 31:18.094 --> 31:21.055 biochemically amount of phosphor a present in 31:21.064 --> 31:22.454 this cultural fibers. 31:22.464 --> 31:23.375 From patient with me, 31:23.515 --> 31:25.925 phosphorylase deficiency was 31:25.935 --> 31:26.645 normal. 31:26.655 --> 31:29.045 Didn't differ from a phosphor a 31:29.055 --> 31:31.464 present in normal culture muscle. 31:31.810 --> 31:34.760 So this was an example of glycogen storage 31:34.770 --> 31:35.579 disease, 31:35.619 --> 31:38.560 which we were not able to reproduce 31:38.569 --> 31:39.500 defect. 31:39.510 --> 31:41.790 We were surprised because my phosphorylase 31:41.800 --> 31:44.579 deficiency was always considered genetically 31:44.589 --> 31:47.469 determined inability of muscle to 31:47.479 --> 31:48.619 produce phosphorylase. 31:48.630 --> 31:49.250 However, 31:49.260 --> 31:51.979 tissue culture studies indicated 31:51.989 --> 31:53.520 that this muscle 31:53.930 --> 31:56.640 growing in Vitra is able to produce 31:56.650 --> 31:56.949 phosphorylase. 31:57.390 --> 31:59.540 And there are probably some other factors 31:59.550 --> 32:01.939 operating in patient which 32:01.949 --> 32:04.469 enable for phosphorylase to be 32:04.800 --> 32:05.819 maintained. 32:08.319 --> 32:11.099 Another example of 32:11.319 --> 32:14.050 glycogen storage disease is 32:14.060 --> 32:16.739 lack of gly of alpha glucosidase, 32:18.180 --> 32:20.760 which is lysosomal enzyme in 32:20.770 --> 32:23.219 another way called acid maltese. 32:23.770 --> 32:26.689 And when acid maltese is absent, 32:26.869 --> 32:29.540 glycogen cannot be break 32:29.550 --> 32:31.189 down to glucose. 32:31.739 --> 32:34.619 And the characteristic feature of this 32:34.630 --> 32:37.410 disease is formation of glycogen 32:37.420 --> 32:40.310 accumulation of glycogen in lysosomal 32:40.329 --> 32:41.199 bound, 32:42.380 --> 32:43.319 we call the sugar. 32:46.180 --> 32:49.030 And uh our first patient 32:49.040 --> 32:51.900 with this disease which ma whose 32:51.910 --> 32:54.609 muscle we have been grown in culture was 32:54.619 --> 32:57.229 32 years old men 32:57.449 --> 33:00.390 who had four years history of 33:00.400 --> 33:03.109 progressive muscle weakness. 33:03.244 --> 33:05.214 He was very healthy till this time. 33:05.224 --> 33:08.185 He was a football player in high 33:08.194 --> 33:11.104 school and he participated in all kinds 33:11.114 --> 33:14.055 of variety of and he was very good in this. 33:14.194 --> 33:14.694 However, 33:14.704 --> 33:16.915 now he developed for the last four years, 33:16.925 --> 33:18.694 proximal muscle weakness 33:20.130 --> 33:22.250 on his biopsy muscle. 33:22.500 --> 33:25.160 There were highly vacated muscle and 33:25.170 --> 33:26.319 both two types. 33:26.329 --> 33:28.939 Type one and type two of fibers were 33:28.949 --> 33:31.579 involved in this cul process. 33:33.250 --> 33:35.760 And then when acid phosphate staining was 33:35.770 --> 33:36.439 performed, 33:36.449 --> 33:39.339 it indicated that these cules 33:39.349 --> 33:41.719 stain positively with acid phosphatase. 33:42.050 --> 33:44.900 What indicated Liz character 33:44.910 --> 33:46.569 of these vales 33:48.479 --> 33:50.489 on the electron microscopy? 33:50.599 --> 33:52.869 We could see this characteristic 33:52.880 --> 33:55.209 appearance of glycogen in 33:55.569 --> 33:57.540 this lysosomal bound 33:58.130 --> 34:00.890 sax membrane bound sacks 34:00.900 --> 34:02.479 here here and here. 34:02.640 --> 34:05.170 And this is characteristic appearance of a 34:05.300 --> 34:08.159 form of acid maltese deficiency 34:08.270 --> 34:10.570 master looks quite normal. 34:10.580 --> 34:13.310 There is no really increase of cytoplasmic glycogen 34:13.689 --> 34:16.070 only accumulation of glycogen in 34:16.080 --> 34:19.060 membrane bound lysosome which we like 34:19.070 --> 34:20.209 to call sugar balls. 34:23.439 --> 34:26.080 When muscle of this patient 34:26.489 --> 34:28.370 were grown in tissue culture, 34:28.379 --> 34:30.179 we were able to see 34:30.629 --> 34:33.320 vacuolization of culture muscle. 34:33.330 --> 34:36.290 After about 10 days in Vitra is 34:36.300 --> 34:38.810 living culture and this is embedded culture 34:39.659 --> 34:40.560 preparation. 34:40.620 --> 34:43.620 We can see vacation when 34:43.629 --> 34:46.050 this vacated area was 34:46.060 --> 34:48.379 selected for electro microscopical 34:48.389 --> 34:49.449 examination. 34:49.909 --> 34:52.540 We could notice even in quite 34:52.550 --> 34:53.800 young fibers, 34:53.810 --> 34:56.010 already some accumulation of 34:56.020 --> 34:58.959 glycogen in this membrane bound lysosome 34:59.389 --> 35:00.610 here and here. 35:00.870 --> 35:01.699 However, 35:01.709 --> 35:04.679 this whole fiber is not very affected yet. 35:04.689 --> 35:07.449 We can see this area is developing has 35:07.459 --> 35:09.320 beginning of zen formation. 35:09.520 --> 35:12.169 There is a lot rougher 35:12.310 --> 35:12.729 the ri. 35:13.729 --> 35:14.600 However, 35:14.610 --> 35:16.590 in ribosomes nice 35:16.600 --> 35:17.760 mitochondria. 35:17.790 --> 35:18.560 However, 35:18.570 --> 35:21.429 already the accumulation of glycogen could be noticed. 35:22.939 --> 35:23.649 However, 35:23.659 --> 35:25.389 when we waited a little bit longer, 35:25.399 --> 35:28.330 we could see that this muscle is becoming 35:28.340 --> 35:29.790 very destroyed. 35:29.830 --> 35:32.570 And all this area is packed 35:32.580 --> 35:35.449 by this glycogen located in 35:35.830 --> 35:38.709 lysosomes here and here here and here which 35:38.719 --> 35:41.469 resemble very much appearance of the 35:41.479 --> 35:43.919 same abnormalities in the spa master biopsy. 35:45.510 --> 35:47.689 And this light illustrates 35:48.060 --> 35:50.689 similarities between patient muscle 35:50.699 --> 35:52.469 biopsy and his culture muscle. 35:52.850 --> 35:55.510 And we can see here there is this 35:55.520 --> 35:58.510 dark membranous membranous material present 35:58.520 --> 36:00.969 in this patient muscle biopsy which could be 36:00.979 --> 36:03.800 detected as well already maybe not 36:03.810 --> 36:06.530 so in pronounced degree in his culture 36:06.540 --> 36:06.899 muscle. 36:06.909 --> 36:08.860 Here and here 36:11.669 --> 36:12.219 again, 36:12.229 --> 36:14.850 doctor from uh Colombia 36:14.860 --> 36:17.030 University Medical Center uh 36:17.050 --> 36:19.340 perform biochemical 36:19.350 --> 36:22.250 examination of this muscle 36:22.260 --> 36:23.879 biopsy in our culture. 36:24.219 --> 36:27.050 And he found that in both this 36:27.060 --> 36:29.830 patient muscle biopsy and in this patient 36:29.840 --> 36:30.260 culture, 36:30.270 --> 36:33.270 muscle acid maltese was missing was 36:33.280 --> 36:35.639 zero as compared to high 36:35.649 --> 36:38.409 amount of acid in the 36:38.419 --> 36:40.080 control muscle biopsy. 36:40.090 --> 36:42.659 And in the culture normal 36:42.669 --> 36:43.370 culture, 36:43.469 --> 36:45.820 this was with natural subs 36:45.830 --> 36:47.409 subtract Marty hydrolysis. 36:48.580 --> 36:51.270 So this case was really our 36:51.280 --> 36:54.229 first example when we were able 36:54.239 --> 36:56.010 to reproduce in Vitra 36:56.709 --> 36:59.689 entirely both morphological and 36:59.699 --> 37:02.639 biochemical defect present in the patient muscle 37:02.649 --> 37:03.340 biopsy. 37:03.510 --> 37:06.000 And we were able in this way to indicate 37:06.010 --> 37:08.699 primary myopathic character of this 37:08.709 --> 37:09.520 disorder. 37:10.020 --> 37:11.639 Later on at this moment, 37:11.649 --> 37:14.030 we are able to grow five 37:14.219 --> 37:17.050 mass muscle from five 37:17.060 --> 37:19.709 patients with acid maltese deficiency and 37:19.719 --> 37:20.449 three heterozygote. 37:22.469 --> 37:24.340 And the this um 37:24.350 --> 37:26.739 picture shows electron 37:26.830 --> 37:28.850 microscopy of 37:28.860 --> 37:31.260 juvenile form of acid maltese 37:31.270 --> 37:32.149 deficiency. 37:32.350 --> 37:35.080 And we can see that there are much more pronounced 37:35.090 --> 37:38.020 abnormalities and even so still this 37:38.899 --> 37:40.770 character of abnormalities, 37:40.780 --> 37:43.239 glycogen bound is 37:43.250 --> 37:45.120 characteristic and present. 37:45.500 --> 37:46.969 There is a lot of glycogen 37:47.350 --> 37:49.280 cytoplasmic as well. 37:49.919 --> 37:52.729 And when this muscle was put to culture to grow, 37:53.239 --> 37:55.030 we have noticed that 37:55.040 --> 37:57.800 vacuolization in these fibers happened 37:57.810 --> 38:00.330 much earlier than in adult form of acid maltese 38:00.340 --> 38:01.120 deficiency. 38:01.320 --> 38:04.199 We could see even very young fibers already are 38:04.209 --> 38:05.209 being vacated. 38:05.219 --> 38:06.879 Even single fibers are being 38:07.129 --> 38:10.120 vacated and on the 38:10.129 --> 38:12.080 electron microscopy as well, 38:12.090 --> 38:14.060 we notice much more 38:14.070 --> 38:16.959 pronounced abnormality than 38:16.969 --> 38:18.209 we have seen in. 38:18.219 --> 38:20.699 From patient with adult form of acid maltese 38:20.709 --> 38:23.500 deficiency biochemical defect in 38:23.510 --> 38:26.179 this juvenile form was reproduced as well. 38:27.149 --> 38:28.080 Last month, 38:28.090 --> 38:30.959 we received muscle biopsy from um 38:30.969 --> 38:33.899 Amsterdam from Doctor Belem from Wilhelm in Augustus 38:33.969 --> 38:34.870 University. 38:35.189 --> 38:37.100 And uh after 20 38:37.110 --> 38:39.050 hours in the plane, 38:39.620 --> 38:42.610 this muscle arrived finally to our laboratory 38:42.689 --> 38:45.590 and we were able to grow this and this 38:45.600 --> 38:47.179 muscle even contracted. 38:48.810 --> 38:51.409 And this was from 38:51.419 --> 38:53.330 patients with biochemically proven, 38:53.340 --> 38:53.750 again, 38:53.760 --> 38:54.570 proven again, 38:54.580 --> 38:56.060 acid maltese deficiency. 38:56.290 --> 38:58.639 And we can see here abandoned nice 38:58.649 --> 39:01.250 growth of culture muscle from this 39:01.260 --> 39:02.649 53 years old plain. 39:03.229 --> 39:03.860 However, 39:03.870 --> 39:06.770 even very young muscle fibers here are already 39:06.780 --> 39:09.719 evacuated and they have glycogen 39:09.850 --> 39:12.699 present and bound in this glycogen 39:12.800 --> 39:14.659 accumulated in lysosomes. 39:15.860 --> 39:18.689 Higher magnification shows this much 39:18.699 --> 39:21.370 better glycogen present in the 39:21.379 --> 39:23.899 membrane bound lysosomes here and 39:23.909 --> 39:26.489 here here in a relatively 39:26.500 --> 39:29.000 young and developing 39:29.090 --> 39:30.060 fiber. 39:32.350 --> 39:33.189 Moreover, 39:33.199 --> 39:36.159 another interesting characteristic which we have never 39:36.169 --> 39:39.070 observed before in cultural muscle was present of 39:39.080 --> 39:41.719 this small sarcoma here 39:41.919 --> 39:44.790 which have never been described in skeletal muscle and we 39:44.800 --> 39:47.489 have never seen this in the skeletal muscle 39:47.500 --> 39:48.120 before. 39:50.399 --> 39:53.290 And here is another fiber from the same 39:53.300 --> 39:53.889 culture, 39:53.899 --> 39:55.899 having this small circo 39:56.419 --> 39:58.330 present there as well. 39:59.090 --> 40:01.879 And the all this that shows 40:01.889 --> 40:04.399 that tissue culture provides 40:04.409 --> 40:07.340 very good system for examination 40:07.350 --> 40:08.620 of muscle pathology. 40:09.219 --> 40:11.659 And we hope that with further 40:11.669 --> 40:14.239 improvement and very exact 40:14.250 --> 40:16.739 morphological electron microscopical and 40:16.750 --> 40:19.629 biochemical examination of cultural human 40:19.639 --> 40:20.120 muscle. 40:20.340 --> 40:22.939 We will be able to learn more and more 40:22.949 --> 40:25.669 about pathogenesis and eventually treatment of 40:25.679 --> 40:27.300 human neuromuscular diseases. 40:27.310 --> 40:27.899 Thank you.