The dream of the stem cell has been fulfilled in an eastern作者：Xu Rongxiang 出版社：CHINA SOCIAL SCIENCES PRESS 发行日期：2009 September
1. To explain MEBT (Moist Exposed Burn Therapy) techniques by empirical theory itself was not enough and satisfactory.
In the early 90s of 20th century, Rongxiang Xu completed his MEBT techniques, which shocked the clinical burn society. His therapeutic techniques had entirely changed the current state of burn treatment. By applying MEBO, his uniquely invented medication, and moist techniques (MEBT), wound healing without scar formation could be achieved even in severe burn cases.
These revolutionary therapeutic effects strongly shocked the traditional burn treatment techniques based on burn surgery. Rongxiang Xu and his medical techniques became the center of debate in traditional burn medicine in China. Some researchers denied the MEBT techniques, blamed Rongxiang Xu as a liar, and his techniques as a scam. On the other hand, other researchers believed that the MEBT techniques were the most advanced achievements in burn medicine. They became advocates and applied MEBT techniques themselves.
Why, among the same group of traditional burn medicine researchers, was there a totally contradicted opinion towards the same technique?
The key question based on which the opponents opposed, or the advocates supported the techniques, was about the mechanisms behind the techniques.
The critics against the moist techniques were based on experiences and traditional theories. In accordance with experiences, with the exception of epidermis that could automatically undergo physiological healing without scar formation after injury, injured deeper skin tissues were not able to heal without scar by themselves. The rationale provided by traditional theory was that the basal stem cells residing in epidermis contributed to epidermal healing after epidermal injury. When the epidermis was injured, basal stem cells were automatically initiated to differentiate into tissues to repair. Even without injury, the old tissues needed to be replaced by new tissues during normal epidermal metabolism, which was carried out by basal stem cells also. Basal stem cells were mostly located in epidermis, with a few found in dermis. As a result, epidermal repair became impossible once the burn was deep into dermis, fat tissues, or underlying muscles where there were no stem cells. Based on this established theory, some researchers confidently concluded: Whatever Rongxiang Xu claimed by his wish that he can cure, it was not real. Nobody can make achievement against the principles of life.
The fact that during the nearly ten years of development, MEBT had successfully treated millions of patients was still not enough to change the opponents’ attitude. Their stubbornness should not be blamed, since the key lay in the fundamental theory. In the time of molecular medicine, the principles of life could be thoroughly and accurately elucidated on molecular level, whereas the approach of moist therapy tried to explain the mechanisms by empirical theory, which was clearly equivocal.
Nevertheless, the supporting researchers, who believed that the fact is the first priority and the theory is only the second, felt the techniques were unfairly treated. The contribution of Rongxiang Xu’s MEBT was not merely raised burn medicine to a level never seen before; more important, it revealed through clinical practice that deep injured skin is able to undergo physiological healing. This was a revolution not only in therapeutic medicine, but also in human recognition. Without moist therapy techniques, our understanding on skin tissue generation would not be intrinsically profound or superficially comprehensive.
MEBT could not be clearly explained in theory, which was not the fault of the techniques. Quite the contrary, it was due to the insufficient understanding by traditional theories in new phenomena, which did not meet the needs in reality. The reality required the tradition to confront the reality, the challenge, and the innovation. Some researchers already sensed the imperative need to commence study on the mechanisms of MEBT techniques and recognized their significance for the future, which will not be limited in burn medicine. The mechanisms would be a ‘nuclear fission’ involving the entire medical field and the future life science. This was likely the reason that the two American researchers, Barbara and Gorden, used ‘Nobel Prize’ as the specification to assess the value of this study.
Rongxiang Xu, the inventor of MEBT techniques, had been diligently strived after the mechanisms behind MEBT for years. After the birth of MEBT, Rongxiang Xu had further improved his techniques, and simultaneously initiated the study on the mechanisms behind skin wound healing without scar. As early as in 1989, on the first issue of the journal The Chinese Journal of Burns Wounds & Surface Ulcers founded by himself, Rongxiang Xu and his research team published their research paper, in which the special cell growth observed under optical microscope was reported. In the last quarter of 1991 the full scale research was ready to launch.
2. Expedition of stem cell army was aborted halfway through
On October 25th, 1991, Barbara, the famous burn specialist from Hackensack University Medical Center informed Rongxiang Xu through international phone conversation about the repeated experiments he performed on BC4D, the core medication of MEBT. The following were the summary of this conversation:
“You visited America in December, 1990. Before leaving, you gave me the study about MEBO. After 1 year efforts, we finally got the results now. By using MEBO you left, we performed following experiments, and the results are satisfying.
The first experiment was to use MEBO to study cell culture in vitro. In short, the experimental methods were as follows: using two groups of epithelial cells in the test tubes all filled with media. MEBO was added into only one group of test tubes (test group). The cell culture results were then compared. The results were remarkable. In the test group, the epithelial cells transformed into mature basal cells with rapid growth, which was determined by precision apparatus. These results, could not be obtained by using any other drug, were a significant world-class breakthrough!
The results of the second experiment: putting dermal explants into cell culture media with MEBO, which resulted in rapid regeneration of residual hair follicle gland epithelium and hair follicle gland under MEBO effects, the morphology of regenerated hair follicle gland and hair follicle appendage were unchanged. All of these were observed directly through microscope. Previously, people had only prefigured the similar change by the same pattern of hair follicle regeneration. However, the growth of hair follicle epithelium was extremely slow so that nobody was able to describe, nor to observe the regeneration directly. But, after MEBO application, considerable increase of the growth rates could be observed directly. This was also a big international breakthrough.
The results of the third experiment regarding molecular biology: we tried to study the ‘regenerative’ gene engineering of regenerated tissue cells applied with MEBO, and measured the DNA and RNA with experimental tools. The experimental results showed: after the application of MEBO, the number of DNA and RNA in the tissue cells were significantly increased, which indicated rapid cellular division… The results, as important as those above, were never attempted and obtained before by anybody in the world.
These results had major impacts on the FDA approval of MEBO entering into American market. Since the experiments were performed in FDA approved laboratory and even funded by FDA, they were totally reliable.”
Even though Rongxiang Xu had completed the experiments performed by Barbara, the information coming across the sea still brought the urgency to him. After the phone conversation, Rongxiang Xu reorganized related files and went to the science department of China’s Ministry of Health. He reported to the persons in charge and suggested to initiate full scale study under government supervision on the mechanisms of moist therapy techniques. His suggestion drew immediate attention from both the department and the ministry.
On October 30th, 1991, directed by the science department of Ministry of Health, a large conference involving researchers in basic medical science from various research authorities, including Chinese Academy of Medical Sciences, Chinese Academy of Preventive Medicine, and Beijing Medical University, was held in Capital Hotel. During the conference, Rongxiang Xu introduced the extraordinary effects of MEBT on physiological regeneration in burn skin tissues. He also recommended the subjects need to be explored further, which included: 1) epidermal regeneration by sweat gland epithelium, 2) full regeneration of dermis and appendages, and 3) the study on the pattern of rapid DNA change.
The report by Rongxiang Xu genuinely encouraged these researchers in basic medical science. Within one month, a detailed study design was prepared and submitted by nearly 50 prominent researchers in basic medical science of China, with up to 150 researchers involved. Facing the thick folders of study description, Rongxiang Xu seemed going back into the ancient world, genuinely felt the greatness of the war of the century like an epic hero.
What a great era, what an encouraging plan, what an exciting study!
While the group study on the pattern of skin cell growth was underway, the battle began and the curtain was rising. Suddenly, Rongxiang Xu was heavily attacked by traditional burn medicine in China.
Though Rongxiang Xu eluded from this adversity by the support from Ministry of Health and the State Council of China, his expeditionary army was dismembered. During the following days, Rongxiang Xu experienced a series of complex and risky events consisted of critics and supports, investigating and being investigated, and frame and counter-frame. He was forced to be involved in a struggle in order to back his MEBT techniques up. The research plan of skin tissue cytology had to be buried deeply in his heart.
Dying before conquer, mourning ever since.
At the end of 1994, Rongxiang Xu’s experience received attention from the newly appointed Health Minister Wenkang Zhang, who inquired to Rongxiang Xu about the research status of the mechanisms in moist therapy techniques. After acknowledging that Rongxiang Xu’s study was terminated due to the academic dispute, Wenkang Zhang resolutely instructed that the study could not be stopped; the Ministry of Health would create a healthy environment to support Rongxiang Xu’s research.
After a cold winter, Rongxiang Xu was welcomed by the warmness of spring. On May 22nd, 1995, he announced the initiation of skin stem cell research on Chinese CCTV Xinwenlianbo (CCTV network news).
3. To locked up ‘induced human keratin 19 positive cells’
Without a large-scale corps, without massive ceremony, Rongxiang Xu, along with his research team with less than 10 people, disappeared from press and public view. On April 1st, 1996, he and his research team left Beijing and headed to Xiangfan, Hubei Province where there happened to be large numbers of severe burn patients receiving the treatment of MEBT techniques. From there, his basic and clinical cytology combined study on skin regenerative mechanisms began.
This time his opinion about the study, comparing with five years ago, had changed dramatically. Despite the time wasted in the struggle for the survival of his study in the last five years, the experience also gave Rongxiang Xu a chance to be composed. From fervor to calmness, the impatience was removed by the time, and the hidden correlation of nature gradually emerged in his mind.
During the five years, Rongxiang Xu had never stopped thinking about the skin regenerative mechanisms. Firstly, these thoughts surfaced as inspirational bursts, full of intellectual sparkles, splattering everywhere, rich and disorderly, just like the wide variety of more than 50 study subjects submitted by other scientists. Gradually, his mind focused on only one subject---stem cell.
Rongxiang Xu’s mind was controlled by a cytological concept of keratin 19 stem cells. Initially, he promptly noticed this cell not through logical deduction, but an inspiration. Nonetheless, he kept himself focusing on this cell and was afraid that it might disappear from his fissile mind. Later, through rational analysis, he predicted even before confirmed by experiments: the basic causative substances for the complete physiological repair of deep injured skin were most likely the keratin 19 positive stem cells. Once the conclusion was drawn, his mind was locked in an unshakable state.
Back in 1989, Rongxiang Xu directly observed through optical microscope that after MEBO application, the cells in the burn tissues underwent specific changes. This was consistent with the in vitro results obtained by Barbara. Once MEBO was applied, large amount of cells proliferated in the burn wound and congregated to form a cell mass. This change could not be seen in the wound without MEBO application. When the tissue slide was sent to Chinese General Hospital of the People's Liberation Army, the authoritative cytologist exclaimed: ‘what is this tissue, how can embryonic tissue cells come to the burn skin wound?!’
Based on the knowledge of skin tissue cells provided by known basic research, Rongxiang Xu acknowledged that the detected skin epidermal cells in various differentiation states contain 5 types of keratin: human keratin 1, 10, 9, 16, and 19. Different cell types have different functional expression. Cells containing positive keratin 1 and keratin 10 are mature epidermal cells with the ability to produce harder stromal cells, their proliferation and differentiation ability are almost exhausted; Keratin 9 and 16 positive cells are immature intermediate cells appeared in the migration process of epidermal stem cells with the ability to differentiate into keratin 1 and 10 positive cells; keratin 19 positive cells are epidermal regeneration cells with the most potential differentiation ability.
All of the above cells contribute to the growth and repair of human skin at different stages. However, except keratin 19 positive cells, all other cells, including keratin 1, 10, 9, and 16 positive cells are intermediate cells naturally residing in epidermis only. Therefore, none of these cells, but keratin 19 positive cells can complete skin regeneration in deep burn wound.
Still, there are mental barriers that make the unquestionable acceptance of keratin 19 more difficult than it should be. Based on what we already know, keratin 19 positive cells are embryonic cells that can only be found in the process of embryo development from zygote in the maternal uterus. By the development of embryo, keratin 19 positive cells will differentiate into other cell types. To identify keratin 19 positive cells, firstly we must find the possible residence, i.e. try to find them in epidermis, dermis, fat layer, and even muscles, where are thought to be impossible for them to reside; in addition, we have to track the alteration and transformation of keratin 19 cells. If we can identify the division, proliferation, and differentiation of keratin 19 cells into keratin 1, 10, 9, and 16 positive cells in the skin healing process in deep burn wound after MEBT, then we can be certain that the keratin 19 cells are found.
4. Conquering stem cell was easy as pie
Theory-first approach made the practice straightforward. Previously it was believed that a full-scale, joint research with multiple subjects was essential to achieve the goal. Now it seems not necessary. The subject was simple and clear: to clarify the cells with keratin 19 positive expression. This enabled a simple research unit to finish the study, which was originally thought a huge project and requiring substantial personnel resources. The research unit was further divided into 2 teams. One was to strictly supervise the clinical practice to make sure that in order to perform basic research observation, patients’ biopsy samples will be accurately obtained as scheduled. The other team was to choose the best laboratory in which the transformation of stem cells would be followed by the most advanced cell tracking technology.
Sometimes destiny counts. Coincidentally, there was a group of severe burn patients in Xiangfan, Hubei Province, which provided a valuable chance for basic stem cell research. Rongxiang Xu himself participated in the rescue and treatment of each patient. Furthermore, he guided researchers to make pathological slides in accordance with the planned specification. At the same time, he had to travel back to Beijing once in a while to discuss experiment designs and methods with researchers from China Medical University. In one occasion, right after arrived at Beijing airport and turn his mobile phone on, he received a phone call from the clinical center in Xiangfan, which informed him that there was a patient in critical situation and the hospital physician was unable to handle. Rongxiang Xu immediately took another flight heading back without hesitancy. Just when the hospital physician decided to give up on that patient, Rongxiang Xu arrived. By his management, the life threatening symptoms in this patient was relieved soon.
There were also setbacks during the experiments in Beijing. Initially, researchers found that the immunofluorescent results showing no keratin 19 positive cells expressed on the slides. Rongxiang Xu was surprised by the results sent to his office. Since he had unbreakable confidence on keratin 19, he did not believe the results. There must be something wrong somewhere. It was hard to find any error through either purely theoretical deduction, or the examination of the experimental procedures applied during the observation. Rongxiang Xu started to analyze the experimental results and found that they were far from normal. The cells on the slides showed mutation generated by passage and were not the original. This indicated that there were errors in the sampling process.
Going back through the sampling methods, Rongxiang Xu found that what he proposed was possibly true. He discussed this with Zenglu Xu, professor of Chinese Academy of Medical Sciences affiliated Peking Union Medical University and a member of study subject validation team, and decided to immediately freeze the slides after sampling and reserve the slides in liquid nitrogen. Once the storage condition was changed, the experimental observation became uncomplicated.
Next step was to perform indirect staining with biotin – avidin DCS system on the biopsy slides. Indirect staining with biotin – avidin DCS system was the most advanced cell staining technique in the world. It could show specific mark through fluorescencent display and therefore, was the ideal cell tracking method.
Firstly, in order to determine the existence of keratin 19 positive cells in the treated burn tissues, researchers selected a monoclonal antibody, mouse anti–human keratin 19 antibody as the tool. During the experiment, the normal skin taken as control and was compared to the burn skin treated by MEBT, to see if there was any specific response. Researchers found no keratin 19 positive cells in the normal skin, which met the anticipation. On the other hand, keratin 19 fluorescent positive cells were found in MEBO treated burn tissues 24 hours after application.
In addition, observation on treated burn tissue cells continued. Four days after treatment, researchers found increased number of keratin 19 cells in sweat gland, capillaries, and the tissues surrounding hair follicle. Seven to fourteen days after treatment, the number of keratin 19 cells reached the peak. Twenty-one to twenty-eight days after treatment, the cell numbers began to drop. These results were consistent with clinical and histological results obtained from treated burn skin.
The experiment achieved the initially established objective to identify keratin 19 positive stem cells, and to track their proliferation and differentiation. The mechanisms of burn skin tissue regeneration and repair emerged.
I. The experiment demonstrated that human keratin 19 positive cells, the most primitive embryonic cells, could be produced in the residual burn tissues; the change of the numbers of keratin 19 positive cells indicated that these cells had the ability to continuously differentiate and proliferate;
II. The results from immunofluorescent stain showed that keratin 19 positive cells could, after migration and division, generated other types of keratin positive (keratin 1 and 10) cells contained in mature epidermis, which sufficiently proved that burn skin tissue regeneration and repair were carried out by these human keratin 19 positive cells;
III. Researchers also primarily confirmed the initiation and regulation model of human keratin 19 positive cells. Research showed that cells were released from stationary phase. The firstly activated protein was cyclin D, which was expressed only by the stimulation of growth factors. The cells in eukaryotic organisms were regulated by cycle protein cyclin D1/CDK4 complex at G1 phase to determine whether to enter proliferative state or withdraw from the cycle. Thus, the initiative and regulatory effects of MEBT techniques on stem cell division and self renewal, and regeneration was proved.
IV. Researchers also discovered that fibroblasts, vascular tissue cells, and skin neural cells were able to continuously proliferate and generate skin by either themselves, or by the interaction between them. The detailed results would be gradually published in the future.
In April, 1998, the research team led by Rongxiang Xu primarily completed the study on identifying the mechanisms in physiological healing of injured skin after treated with MEBT/MEBO. The beautiful dream of treating clinical diseases with stem cells was fulfilled in the ancient east.