研究人员转换肝硬化引起细胞健康的肝细胞在小鼠体内

StephenW

            Researchers convert cirrhosis-causing cells to healthy liver cells in mice                            June 2, 2016                                                                                                        
        UCSF researchers have learned to generate healthy new liver cells (dark green, right side) within the livers of living mice by converting the very cells that drive liver disease (red, left side), thereby reducing liver damage and improving liver function at the same time. Credit: Willenbring lab / UCSF        Advances in stem cell research have made it possible to convert patients' skin cells into heart cells, kidney cells, liver cells and more in the lab dish, giving researchers hope that one day such cells could replace organ transplantation for patients with organ failure. But successfully grafting these cells into patients' failing organs remains a major clinical challenge.             

                               Now a team of researchers led by UC San Francisco scientists has demonstrated in mice that it is possible to generate healthy new liver cells within the organ itself, making engraftment unnecessary. What's more, they did it by converting the very cells that drive liver disease, thereby reducing liver damage and improving liver function at the same time. The technique takes advantage of a viral gene delivery technology that has gone through early validation in patients for liver-directed gene therapies, suggesting it could be readily translated into a therapy for patients with liver disease, said Holger Willenbring, MD, PhD, a professor of surgery at UCSF and senior author of the new study, published June 2, 2016 in the journal Cell Stem Cell.
"Part of why this works is that the liver is a naturally regenerative organ, so it can deal with new cells very well. What we see is that the converted cells are not only functionally integrated in the liver tissue, but also divide and expand, leading to patches of new liver tissue," said Willenbring, who is also associate director of the Liver Center at UCSF and a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research.
More than 600,000 patients in the United States alone suffer from end-stage liver disease, or cirrhosis. The only available cure is liver transplantation, but the shortage of donor livers means only 6,000 patients benefit from this therapy each year in the U.S., and more than 35,000 patients die.
The new approach specifically targets liver fibrosis, the progressive scarring of the liver that is a primary driver of liver disease. Fibrosis develops when liver cells called hepatocytes can't regenerate fast enough to keep up with damage caused by toxins such as alcohol or diseases such as hepatitis C or fatty liver disease. Cells called myofibroblasts fill in gaps left by dying hepatocytes with scar-like fibrotic tissue. It's a bit like patching a flat tire: at first the patches help maintain the liver's structural integrity, but eventually a liver that is more patches than functional tissue starts to fail.
                
                   Fibrotic liver disease has a slow fuse, but leads to catastrophic failure: someone who contracts hepatitis C at age 25 may feel just fine for decades, then suddenly at age 50 start experiencing the fatigue, faintness, nausea, bruising, diarrhea and jaundice that indicate the onset of end-stage liver disease. The reason is that the liver can adapt as long as at least 20 percent of it is functional, Willenbring says, but once it dips below that critical threshold, patients are often dead within two years.
"Liver fibrosis is not rare," Willenbring said. "It's actually the end stage of many chronic liver diseases. Obesity, for instance, can lead to fatty liver disease, which is predicted to become the number one cause of liver fibrosis in the next 10 years."
Technique reduces liver damage, increases liver function in mice
Willenbring has dedicated his career to using stem cell biology to generate new hepatocytes (for instance, from a patient's own skin) that could be used in place of transplants to treat liver failure.
Ironically, however, just as he and his lab have come closer to achieving this dream, Willenbring has had to grapple with the realization that this cell therapy approach may fail to reach the majority of patients with liver failure because the tangle of fibrosis itself impairs the successful engraftment of transplanted cells.
So over the past five years, Willenbring and his team—in close collaboration with the laboratory of Dirk Grimm, PhD at Heidelberg University Hospital in Germany—have embarked on a different approach: converting fibrosis-causing myofibroblasts into healthy new hepatocytes within the liver itself.
Previous research had identified a cocktail of gene-regulating proteins that can convert other cell types into hepatocytes, but the team needed a way to deliver these instructions to myofibroblasts. After several years of work, the team identified a subtype of adeno-associated virus (AAV) that could specifically infect myofibroblasts. The researchers focused on AAV because it has been shown to be safe and effective in early human gene therapy trials, e.g., for therapy of the bleeding disorder hemophilia B.
The team showed in mice with liver disease that viruses packed with the cell fate-changing cocktail indeed infected myofibroblasts and converted them into functional hepatocytes. The number of new cells was relatively small - less than one percent of all hepatocytes in the treated mice - but this was sufficient to reduce fibrosis and improve liver function.
The viral approach was also effective in converting human myofibroblasts in a dish into working hepatocytes, but more work is certainly needed to prepare this approach for use in human patients, the researchers say. In particular, the lab is working to package the treatment into a single virus, reducing potential side effects and streamlining clinical development. The team is also working to make the viruses more specific to myofibroblasts - in the current paper muscle cells and some cells of the immune system were also infected, though without converting them into liver cells or obviously impacting their function.
The new results suggest that in the fibrotic liver this approach could produce a more efficient and stable improvement of liver function than cell transplant approaches, Willenbring said, adding that once the viral packaging is optimized, such a treatment could be done cheaply at a broad range of medical facilities, not just in the specialized research hospitals where stem-cell transplants could be conducted.
Willenbring is the first to acknowledge that the new technique is not a replacement for the current standard of care in liver disease: "A liver transplant is still the best cure," Willenbring said. "This is more of a patch. But if it can boost liver function by just a couple percent, that can hopefully keep patients' liver function over that critical threshold, and that could translate to decades more of life."                         

               More information: Cell Stem Cell, DOI: 10.1016/j.stem.2016.05.005
                                        Provided by: University of California, San Francisco
                   

StephenW

研究人员转换肝硬化引起细胞健康的肝细胞在小鼠体内
2016年6月2日
研究人员转换肝硬化引起细胞健康的肝细胞在小鼠体内
加州大学旧金山分校的研究人员已经了解到通过转换驱动肝病（红，左侧）的非常细胞，从而减少了肝损伤和改善肝功能的同时，以产生活小鼠的肝脏内健康的新肝细胞（深绿色，右边）时间。信用：韦兰博凌实验室/加州大学旧金山分校

在干细胞研究进展使人们有可能对病人的皮肤细胞转化成心脏细胞，肾细胞，肝细胞等在实验室培养皿，让研究人员希望有一天这种细胞可以替代器官移植患者的器官功能衰竭。但是，成功地移植这些细胞进入患者的失败机关仍然是一个主要的临床挑战。

现在一个队由加州大学旧金山分校的科学家领导的研究已经证明在小鼠中，这是可能的器官本身内产生健康的新肝细胞，使得植入不必要的。更重要的是，它们在同一时刻转换驱动肝病的非常细胞，从而减少了肝损伤和改善肝功能做到了。该技术采用的病毒基因递送技术已经通过早期验证了患者的肝脏定向基因疗法的优势，这意味着它可以很容易地转化为肝病患者的治疗方法，霍尔格韦兰博凌，博士，教授说：在加州大学旧金山分校的手术和新的研究发表2016年6月2日的Cell杂志干细胞的资深作者。

“为什么这个作品部分是肝脏是一个自然再生的器官，所以它可以处理新的细胞非常好。我们所看到的是，转换后的细胞不仅在功能上集成在肝组织，而且分裂和扩大，导致新肝组织的补丁，“韦兰博凌，谁也与肝病中心在加州大学旧金山分校的副主任的礼和再生医学的Edythe远大中心的成员和干细胞研究说。

60多万患者在美国就有患有终末期肝病或肝硬化。唯一可用的治疗是肝移植，但供肝短缺意味着只有6000例患者从这种疗法每年受惠于美国，并且超过35,000例患者死亡。

的新方法特别针对肝纤维化，即肝脏疾病的主要驱动肝脏的渐进疤痕。纤维化发展时称为肝细胞的肝细胞不能再生速度不够快，以跟上毒素引起的诸如醇或疾病例如丙型肝炎或脂肪肝疾病的损害。细胞称为肌纤维母细胞填入与瘢痕样纤维组织死于肝细胞留下的空白。这是一个有点像修补爆胎：首先补丁帮助维持肝脏的结构完整，但最终肝脏是更多的补丁比功能组织开始失败。

肝纤维化疾病有一个缓慢的导火索，但会导致灾难性的失败：有人谁在25岁时患丙肝可能觉得蛮好了几十年，突然在50岁开始经历了疲劳，头晕，恶心，青紫，腹泻及黄疸指示终末期肝病的发病。其原因是，肝脏可以，只要它至少有20％是功能适应，韦兰博凌说，但一旦它下降到低于该临界阈值，患者往往两年内死亡。

“肝纤维化是并不少见，”韦兰博凌说。 “它实际上是许多慢性肝病的终末阶段，肥胖，例如，可导致脂肪肝疾病，预计将成为肝纤维化的头号原因，在未来10年。”

技术减少肝损伤，小鼠增加肝功能

韦兰博凌一直致力于他的职业生涯是利用干细胞生物学产生新的肝细胞（例如，从患者自己的皮肤），可以代替移植可用于治疗肝功能衰竭。

讽刺的是，然而，就在他和他的实验室已经进一步实现这个梦想，韦兰博凌已与实现搏斗，这种细胞治疗的方法可能会失败，肝功能衰竭，达到广大患者由于肝纤维化的纠结本身损害移植细胞的成功植入。

因此，在过去的五年中，韦兰博凌和他的团队，在德克格林，在德国已经走上了不同的方法博士在海德堡大学医院的实验室密切合作：转换纤维化导致肌纤维母细胞到健康的新肝细胞肝癌本身。

以前的研究已经确定基因调控蛋白，可以转换其他类型的细胞为肝细胞的鸡尾酒，但球队需要一种方式来提供这些说明，肌纤维母细胞。经过几年的工作，该小组确定腺相关病毒（AAV），可以特异性感染肌纤维母细胞的一个亚型。的研究人员集中研究的AAV，因为它已被证明是安全和有效的早期人类基因治疗试验中，例如，对于出血性疾病血友病B的治疗

球队在肝脏疾病的小鼠，病毒挤满了细胞命运变化的鸡尾酒确实感染肌成纤维细胞表现出并将它们改建肝细胞的功能。新细胞的数量相对较小 - 小于在处理的小鼠中所有的肝细胞的百分之一 - 但这足以减少纤维化和改善肝功能。

研究人员说，病毒的方法也有效地转化成纤维细胞的人类在培养皿中进入工作肝细胞，但更多的工作肯定是需要准备这种方法用于人类患者使用。具体地说，实验室工作打包处理成一个单一的病毒，从而减少潜在的副作用和精简临床开发。该小组还努力使病毒更具体的肌纤维母细胞 - 在当前纸张肌肉细胞和人也感染免疫系统的一些细胞中，虽然没有将其转化为肝细胞或明显影响其功能。

新的研究结果表明，在肝纤维化这种方法可产生肝功能比细胞移植的方法的一种更有效的和稳定的改善，韦兰博凌补充说，一旦病毒包装被优化，这样的治疗可以廉价地在大范围进行医疗设施，不仅在专业研究的医院在哪里干细胞移植可同时进行。

韦兰博凌是第一个承认新技术也并非照顾肝病当前标准的替代品：“一个肝脏移植仍是最好的治疗，”韦兰博凌说。 “这更像是一个补丁，但如果它可以通过短短几个百分比提高肝功能，能够保持希望患者的肝功能超过了临界值，这可能转化为更多数十年的生活。”

更多信息：细胞干细胞，DOI：10.1016 / j.stem.2016.05.005

加州大学旧金山分校：由提供