Combining iPSC and gene editing with cell therapy to develop the next generation of MSC-based therapeutics to counteract bone fragility in OI.

骨质流失和骨骼脆弱造成了巨大的医疗保健和经济负担，由于衰老过程、微重力、长时间固定或骨质疏松症或脆性骨病成骨不全症（OI）等病理，影响了世界上数百万人。成骨不全是由于遗传缺陷导致骨基质的主要成分（I型胶原蛋白）出现缺陷，阻碍了成骨细胞（成骨细胞）的成熟，从而导致骨骼变脆。成骨不全无法治愈，患者一生都需要护理。因此，开发治疗方法来对抗骨骼脆弱是至关重要的。利用成骨不全的临床前模型，我们最近表明，从健康婴儿周围的妊娠液中分离的人间充质干细胞（MSC）移植改善了脆弱骨骼的质量，刺激了常驻成骨细胞的成熟，并降低了常驻骨吸收细胞（破骨细胞）的活性。然而，在骨髓间充质干细胞常规应用于临床之前，有两个主要障碍需要克服。首先，从供体器官或妊娠液中分离的间充质干细胞在实验室培养达到足够数量时逐渐失去其再生潜力。其次，我们需要了解MSC如何在体内发挥作用，以改进移植方案，并开发测试来确定假定的新供体细胞样本的修复潜力。幸运的是，MSC的新来源是再生细胞，也称为诱导多能干细胞（iPSC）。从健康或成骨不全患者身上分离出来的皮肤细胞可以在体外恢复活力，导致成骨不全的遗传缺陷可以通过基因组编辑修复。由于iPSC在培养过程中不会发生变化，因此可以在将其转化为MSC之前获得足够数量的细胞。这些细胞被称为诱导间充质干细胞（iMSC）。这提高了利用来自患者自身细胞的iMSC开发下一代细胞疗法的可能性。该项目的目的是测试iMSC在对抗骨脆弱性和替代原发MSC使用方面的潜力，并了解供体MSC如何改善成骨不全。该项目将测试iMSC（来自健康的iPSC或来自纠正遗传缺陷后的OI-iPSC）移植到成骨不全症临床前模型中是否能改善骨质量并使成骨细胞功能正常化。我们还将确定供体间充质干细胞向成骨细胞谱系的分化是否直接有助于骨形成和/或间充质干细胞释放的小袋（称为外泌体）是否具有激活剂的功能，以刺激常驻成骨细胞和/或抑制破骨细胞。最后，我们将使用体外共培养来评估成骨不全遗传缺陷如何阻碍成骨不全人类和小鼠成骨细胞的行为，并确定骨形成和骨吸收细胞的功能是否可以通过人类间充质干细胞通过直接细胞接触，或通过外泌体或人类间充质干细胞分泌的可溶性蛋白来调节。我们的长期目标是利用患者自身的皮肤细胞，为患有骨质流失和有骨折风险的人开发个性化的治疗方法。

Bone loss and skeletal fragility impose a huge healthcare and economic burden, affecting millions of people in the world due to the ageing process, microgravity, prolonged immobilisation, or pathologies such as osteoporosis or the brittle bone disease osteogenesis imperfecta (OI). OI develops because a genetic defect causes the main component of the bone matrix (type I collagen) to be faulty, preventing the bone-forming cells (osteoblasts) from maturing, which causes the bones to become brittle. There is no cure for OI and patients need care all their lives. Thus, developing therapeutics to counteract bone fragility is of paramount importance. Using a pre-clinical model of OI, we have recently shown that transplantation of human mesenchymal stem cells (MSC) isolated from the pregnancy fluid surrounding healthy babies improved the quality of fragile bones, stimulated the maturation of resident osteoblasts and reduced the activity of resident bone resorbing cells (osteoclasts). However, there are two main hurdles to overcome before MSC can be routinely used in the clinic. First, MSC isolated from donor organs or pregnancy fluid progressively lose their regenerative potential whilst they are cultivated in the laboratory to reach sufficient numbers. Second, we need to understand how MSC work in vivo to improve transplantation protocols and develop tests to determine the repair potential of putative new donor cell samples. Luckily, a new source of MSC is rejuvenated cells, also called induced pluripotent stem cells (iPSC). Skin cells, which can be isolated from healthy or OI patients, can be rejuvenated in vitro and the genetic defect causing OI can be repaired through genome editing. Since iPSC do not change during culture, it is possible to obtain sufficient number of cells before converting them back into MSC. These cells are called induced MSC (iMSC). This raises the possibility to develop the next generation of cell therapy using iMSC derived from the patient's own cells. The aim of this project is to to test the potential of iMSC to counteract bone fragility and replace the use of primary MSC and also to understand how donor MSC ameliorate OI bones. This project will test whether transplantation of iMSC (derived from healthy iPSC or from OI-iPSC following correction of the genetic defect) into a pre-clinical model of OI improves bone quality and normalise osteoblast function. We will also establish whether the differentiation of donor MSC towards the osteoblast lineage directly contributes to bone formation and/or whether the tiny sacks (called exosomes) released by MSC function as activators to stimulate resident osteoblasts and/or inhibit osteoclasts. Finally, we will use ex vivo co-culture to assess how the OI genetic defect impedes the behaviour of OI human and mouse osteoblasts and determine whether the function of bone-forming and bone-resorbing cells can be modulated by human MSC through direct cell contact, or via exosomes or the soluble proteins secreted by human MSC. Our long-term goal is to develop personalized treatments for people suffering from bone loss and at risk of fractures using patients' own skin cells.