Homing of MSCs in animal models
Because MSCs may be used for tissue repair, it is of interest to know into which organs MSCs home. Allogeneic and autologous MSCs distributed to a wide range of tissues in baboons (Fig. 3) . Radiolabelled MSCs injected intravenously in rats showed high radioactivity first in the lung and thereafter in the liver . Low radioactivity was demonstrated in kidneys, spleen and bones. Similarly, human MSCs were shown to engraft in multiple tissues and demonstrate site-specific differentiation after intrauterine transplantation into sheep [25, 91]. In a mouse model of osteogenesis imperfecta, MSCs expressing normal type 1 collagen were infused, engrafted and normal collagen was detected . In healthy experimental animals, MSCs home to most organs.
Figure 3. Detection of MSCs after intravenous injection in (a) experimental animals and (b) humans. MSCs transduced with green fluorescent protein distributed to a wide range of tissues, including intestine, pancreas, liver, lung, kidney, urethra, spleen, lymph node, thymus, skin, cerebellum, etc. following systemic infusion into nonhuman primates . Gastrointestinal tissues harboured high concentrations of transgene per microgram of DNA. Kidney, lung, thymus and skin also contained high amounts of DNA. Engraftment ranged from 0.1% to 2.7%. Radiolabelled MSCs injected i.v. in rats showed high radioactivity first in the lung and thereafter in the liver . After i.v. infusion, human MSCs were detected in the circulation in some, but not all, patients within the first hour of infusion, not thereafter . Engraftment of MSCs was demonstrated in five of six patients with severe osteogenesis imperfecta at one or more sites, including bone, skin and marrow stroma . MSC donor DNA was detected in the colon and lymph node at autopsy in a patient treated with MSCs for steroid-resistant GVHD .
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Clinical studies using human MSCs
Mesenchymal stem cells have been brought into the clinic for several purposes: to differentiate and heal damaged tissues, to promote haematopoietic engraftment after transplant through the secretion of growth factors and for immunosuppression in GVHD. As the immunomodulatory mechanisms differ, for instance, between murine and human MSCs, animal models may not predict the clinical situation. For fear of adverse effects, including ectopic tissue formation and tumour development, severe cases were first considered for treatment. Two feasibility studies were performed by intravenous infusion of autologous human MSCs with no adverse reactions observed [93, 94] (Table 1).
Table 1. In vivo experience of human mesenchymal stem cells (MSCs)
|Disease||Number of cases||Source of MSCs||Outcome||Detection of MSCs||Reference|
|Haematological malignancies||15||Autologous||No adverse events||No||Lazarus et al. |
|Breast cancer||28||Autologous||Safe, no side effects||Yes||Koçet al. |
|Inborn errors of metabolism||11||HLA-identical||No immune response against donor||No||Koçet al. |
|Osteogenesis imperfecta||3||HLA-identical||New dense bone formation; few fractures||Yes||Horwitz et al. |
|Osteogenesis imperfecta||6||HLA-identical||Engraftment of gene-marked MSCs||Yes||Horwitz et al. |
|Osteogenesis imperfecta||1||HLA-mismatched fetal MSC||Regularly arranged bone; few fractures Engraftment of mismatched MSCs||Yes||Le Blanc et al. |
|Acute myeloid leukaemia||1||HLA-haploidentical MSCs||Haematopoietic engraftment with no GVHD||No||Lee et al. |
|Leukaemia, aplastic anaemia, SCID*||7||HLA-identical or haploidentical||Enhanced engraftment of HSCT||No||Le Blanc et al. |
|Severe aplastic anaemia||1||Allogeneic MSCs||Engraftment, improved stroma||Yes||Fouillard et al. |
|Severe acute GVHD||1||Haploidentical MSCs twice||Clearance of grade IV acute GVHD,||Possible||Le Blanc et al. |
|Hypophosphatasia, Hunter, vasculitis||3||Allogeneic bone fragments||Stroma cell engraftment||Yes||Cahill et al. |
|Leukaemia||46||HLA-identical||Safe, no side effects||Yes||Lazarus et al. |
|Malignancies, severe acute GVHD||9||HLA-identical, haploidentical and mismatched||Complete response of severe acute GVHD in 6/8||Yes||Ringdén et al. |
|Malignancies, tissue toxicity||10||HLA-identical, haploidentical and mismatched||Resolution of haemorrhagic cystitis, pneumomediastinum and colon perforation||Yes||Ringdén et al. |
Mesenchymal stem cells express high levels of arylsulphatase A and α-l-iduronidase . The deficiency of these enzymes leads to failure to hydrolyse a distinct substrate, leading to its accumulation and dysfunction of multiple organs, the most severe being mental retardation. Arylsulphatase A deficiency is the cause of metachromatic leukodystrophy, and α-l-iduronidase deficiency is the cause of Hurler’s disease, disorders that may be prevented by allogeneic HSCT, which is the only potential cure [96, 97]. MSCs were expanded in vitro and given intravenously to patients with metachromatic leukodystrophy and Hurler’s disease, who had previously undergone HSCT to potentiate and enhance enzyme production in patients who still had some symptomatic disease after transplant . In four of five patients with metachromatic leukodystrophy, there was clear evidence of improvement in nerve conduction velocity.
Mesenchymal stem cells may be used to treat bone disorders such as osteogenesis imperfecta . Five patients with osteogenesis imperfecta treated with bone marrow transplantation had donor osteoblast engraftment, new dense bone formation, an increase in total bone mineral content, increases in growth velocity and reduced frequencies of bone fractures [100, 101]. This suggests that HSCT leads to engraftment of functional MSCs. Gene-marked MSCs, in order to identify the cells after infusion, were given to six children who had undergone HSCT for severe osteogenesis imperfecta . Engraftment of MSCs in the bone and an acceleration of growth velocity were demonstrated. We performed in utero transplantation of male fetal HLA-mismatched MSCs to a female fetus with bilateral intrauterine femur fractures, diagnosed with severe osteogenesis imperfecta . A bone marrow biopsy showed 0.3–7.4% Y-chromosome-positive cells by fluorescent in situ hybridization, indicating engraftment of the donor MSCs. The bone was regularly arranged, with configured bone trabecula lined by a columnar layer of normal osteoblasts. The patient has had fewer fractures than expected for a child with severe osteogenesis imperfecta.
Lee et al. reported a patient with acute leukaemia who received a peripheral blood stem cell graft together with MSCs from her HLA-haploidentical father treated with standard immunosuppression . The patient had rapid engraftment with no acute or chronic GVHD and was well 31 months after transplantation. With conventional immunosuppression and a haploidentical nonmanipulated graft, the risk of rejection or severe GVHD is extremely high. MSCs were given in a pilot study together with HSCT in seven patients to enhance engraftment . In three of these patients, MSCs were given because of previous graft failure and re-transplantation. MSCs were given at a dose of 1 × 106/kg and were HLA-identical in three cases and haploidentical in four cases. Neutrophils >0.5 × 109/L and platelets >30 × 109/L were achieved at a median of 12 days. All patients had 100% donor chimerism within 100 days. In one patient, Henoch-Schönlein purpura resolved. This study encourages controlled trials to use MSCs to enhance engraftment after HSCT.
An elderly woman with end-stage severe aplastic anaemia, suffering from pancytopenia with haemorrhages and infections, received MSCs derived from her HLA-haploidentical son on two occasions . Engraftment of donor MSCs was detected by donor chimerism using polymerase chain reaction showing MSCs in the endosteum of a bone marrow biopsy specimen, but not in bone marrow aspirates. This is also in keeping with studies in baboons and rodents indicating that transplanted MSCs are located in the bone tissue rather than in the marrow cavity, and can be detected in bone biopsies but not in marrow aspirates [10, 105]. At the same time as HSCT, 46 patients received culture-expanded MSCs from their HLA-identical sibling donors . MSC infusions were well tolerated [93, 106]. Moderate to severe acute GVHD was observed in 13 (28%) of 46 patients. Chronic GVHD was observed in 22 (61%) of 36 patients and 2-year progression-free survival was 53%. No MSC-associated toxicities were seen. Stromal cell chimerism was demonstrated in 2 of 19 examined patients at 6 and 18 months after transplantation. From this we can learn that MSCs are safe to give, but are difficult to detect after infusion, even in immunocompromised patients who have undergone HSCT.
At the same time as HSCT, three children with potentially fatal diseases, such as hypophosphatasia, Hunter’s disease and vasculitis, had bone fragments implanted intraperitoneally and into bone . Donor osteoblast-like cells were also infused intravenously after transplant. Chimerism analysis of bone biopsies showed 25–60% donor stromal cell engraftment. This study shows that by bone implantation, donor stromal cells engraft.
We transplanted haploidentical MSCs to a patient with severe treatment-resistant grade IV acute GVHD of the gut and liver . The aim was to use the tissue repair effect shown in vivo in animal models, and the immunomodulatory effects seen in vitro on human lymphocytes. The clinical response was striking with normalization of stool and bilirubin on two occasions, with voluminous haemorrhagic diarrhoea and highly elevated bilirubin being the most pronounced signs of GVHD in this patient. Subsequently, we summarized our experience of treating eight patients with steroid-refractory grades III–IV acute GVHD and one patient with extensive chronic GVHD . Acute GVHD disappeared completely in six of eight patients and the survival curve was better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSCs (P = 0.03). These studies have been extended and in Europe, 55 patients have been treated for steroid-resistant acute GVHD with an overall response rate of 69% . Nonresponders have died of progressive GVHD and several responders have died from infections with an overall survival of 23 of 55 (42%) from 2 months to 5 years. Although the experience is limited, MSCs seems a promising treatment for severe steroid-resistant acute GVHD.
Ten patients undergoing HSCT were treated with MSCs due to tissue toxicity . In five patients, severe haemorrhagic cystitis cleared after MSC infusion. Gross haematuria disappeared after a median of 3 (1–14) days. Two patients with grade 5 haemorrhagic cystitis had reduced transfusion requirements after MSC infusions, but both died of multi-organ failure. MSC donor DNA was demonstrated in the urinary bladder in one of them. Two patients were treated for pneumomediastinum, which disappeared after MSC infusion. A patient with steroid-resistant GVHD of the gut experienced perforated diverticulitis and peritonitis that was dramatically reversed twice after infusion of MSCs. These preliminary data suggest that MSCs may also play a role in repairing severe tissue toxicity.