Mesenchymal stem cells

Human mesenchymal stem cells (MSCs) represent a mesodermal-derived population of multipotent stromal cells. Friedenstein first described the MSCs in the 1960s observing that non-haematopoietic cells which adhered to plastic could be isolated from adult bone marrow , but they were not fully characterised until 1999 where strict culture techniques demonstrated their expansion and multilineage potential in culture . Adult bone-marrow-derived MSCs remain the best characterised source of human MSCs to date, but considerable research has also been performed on MSCs isolated from other sources and developmental stages, many of which are described in this book.

MSC during the development

MSCs make up only a minor fraction of tissues and are, for example, about 10-fold less abundant than haematopoietic stem cells (HSCs) in the bone marrow, but they are easily isolated and propagated in vitro. The prevalence of MSCs declines with advancing age: In the marrow of a newborn, one MSC is found among 10,000 nucleated bone marrow cells, compared with one MSC per 250,000 nucleated cells in adult bone marrow and one per 2 × 10 ⁶ in that of an 80-year-old . By contrast, the foetus is relatively rich in MSCs. It has been shown that the first-trimester foetal blood contains one MSC among every 3000 nucleated cells and the second-trimester foetal bone marrow one MSC among every 400 cells . Human foetal MSCs are present in the foetal circulation from early gestation and then progressively decline in frequency in foetal blood during the late first trimester, suggesting that they may play an important role in establishing foetal haematopoiesis. One hypothesis is that foetal MSCs migrate to the definitive site of haematopoiesis in the bone marrow where they adhere and act as stromal support to the HSCs. This function by the MSCs is supported by the detection of maximal numbers of fibroblast colony-forming units in murine foetal liver, spleen and bone marrow at the time haematopoiesis begins at each site, suggesting the existence of a stromal stem cell population migration on which HSCs are seeded . Foetal MSCs have been demonstrated to localise within haematopoietic sites throughout ontogeny, consistent with parallel and coordinated development of the haematopoietic and mesodermal systems . In line with this, it has been determined that foetal MSCs support haematopoiesis in long-term cultures and maintain expansion of umbilical cord blood-derived HSC in vitro , and animal studies show that foetal MSCs support and enhance engraftment of HSC . These data indicate that the foetal MSCs are implicated in the establishment of haematopoiesis.

MSC during the development

MSCs make up only a minor fraction of tissues and are, for example, about 10-fold less abundant than haematopoietic stem cells (HSCs) in the bone marrow, but they are easily isolated and propagated in vitro. The prevalence of MSCs declines with advancing age: In the marrow of a newborn, one MSC is found among 10,000 nucleated bone marrow cells, compared with one MSC per 250,000 nucleated cells in adult bone marrow and one per 2 × 10 ⁶ in that of an 80-year-old . By contrast, the foetus is relatively rich in MSCs. It has been shown that the first-trimester foetal blood contains one MSC among every 3000 nucleated cells and the second-trimester foetal bone marrow one MSC among every 400 cells . Human foetal MSCs are present in the foetal circulation from early gestation and then progressively decline in frequency in foetal blood during the late first trimester, suggesting that they may play an important role in establishing foetal haematopoiesis. One hypothesis is that foetal MSCs migrate to the definitive site of haematopoiesis in the bone marrow where they adhere and act as stromal support to the HSCs. This function by the MSCs is supported by the detection of maximal numbers of fibroblast colony-forming units in murine foetal liver, spleen and bone marrow at the time haematopoiesis begins at each site, suggesting the existence of a stromal stem cell population migration on which HSCs are seeded . Foetal MSCs have been demonstrated to localise within haematopoietic sites throughout ontogeny, consistent with parallel and coordinated development of the haematopoietic and mesodermal systems . In line with this, it has been determined that foetal MSCs support haematopoiesis in long-term cultures and maintain expansion of umbilical cord blood-derived HSC in vitro , and animal studies show that foetal MSCs support and enhance engraftment of HSC . These data indicate that the foetal MSCs are implicated in the establishment of haematopoiesis.

Phenotype of MSC

Foetal MSCs have similar characteristics as adult bone marrow-derived MSCs; they grow as spindle-shaped fibroblastic cells displaying colony-forming capacity in low-density cultures . In 2006, the International Society for Cellular Therapy stated the minimal criteria for MSCs as follows: adherence to plastic; positive for cluster of differentiation (CD)105, CD73 and CD90 and negative for haematopoietic and endothelial markers and co-stimulatory molecules CD45, CD34, CD14 or CD11b, CD79a or CD19 and human leucocyte antigen (HLA-DR); and ability of tri-lineage differentiation into bone, fat and cartilage . There is no identified specific surface marker for MSC, but the cells are positive for a wide range of surface markers such as CD29, CD44, CD146, CD166 and CD271 . MSCs also express a variety of cell adhesion molecules as integrins α1, α2, α3, α4, α5, α6, αv, β1, β3, β4 and β5 Further characterisation shows expression of ligands for surface proteins present on cells of the haematopoietic lineage, including intercellular adhesion molecule (ICAM)-1, ICAM-2, vascular cell adhesion molecule (VCAM)-1, lymphocyte function-associated antigen (LFA3), CD72 and C-X-C chemokine receptor type 4 (CXCR4) , molecules important in cell binding and homing interactions. Several extracellular matrix molecules such as collagen, fibronectin, laminin and proteoglycans are also secreted by MSCs, suggesting that they may play a central role in the organisation of the extracellular matrix . In addition, MSC constitutively secrete an extensive array of factors and micro vesicles that may act in a paracrine fashion in vivo .

Stem cell properties of MSCs

MSCs are defined as multipotent cells, which means that they are capable of differentiating into mesodermally derived tissues such as bone, cartilage, tendon, bone marrow stroma and adipose tissue. Single-cell clones of expanded foetal and adult MSCs retain their multilineage potential , and the single-cell colonies co-express gene characteristics for osteoblastic, chondrocytic, adipocytic, myoblast, haematopoiesis-supporting stromal, endothelial, epithelial and neuronal lineages . Furthermore, in immunocompetent allogeneic/xenogeneic animal models, it has been shown that when infused intravenously, MSCs engraft widely in multiple tissues and demonstrate site-specific differentiation .

Immunological properties of MSCs

Human MSCs are generally considered to be non-immunogenic in nature. Human foetal MSCs express intermediate levels of HLA class I antigens and do not express class II antigens Foetal MSCs, like adult MSCs, escape recognition by the immune defence in vitro suggesting that they are not inherently immunogenic . Neither foetal nor adult MSCs are killed by allogeneic-naïve natural killer (NK) cells, but when co-cultured with interleukin (IL)-2-activated NK cells, foetal MSCs are killed to a higher extant compared with adult MSCs . Furthermore, they are killed via different pathways; foetal MSCs preferably via the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway and adult MSCs via the Fas ligand (FasL) pathway . Lastly, foetal MSCs possess immunomodulatory properties as they inhibit mitogen-induced allogeneic lymphocyte proliferation .

Differences between foetal and adult MSCs

Some differences exist between foetal and adult MSCs. Foetal MSCs are more primitive cells and are found at a higher frequency possessing greater colony-forming capacity in comparison to adult MSCs. First-trimester foetal MSCs demonstrated extensive proliferative capacity and cycle faster compared with adult MSCs, having a doubling time of 30 h versus 80 h and attained 28.4 versus 7.1 population doublings over 50 days, respectively . Foetal MSCs can be expanded for up to 70 population doublings without signs of senescence or apoptosis, whereas adult MSCs typically undergo about 15–40 cell doublings in vitro before senescing . Consistent with this, foetal MSCs have increased expression of telomerase and have longer telomeres as well as increased expression of transcripts implicated in cell-cycle promotion, chromatin regulation and DNA repair compared with adult MSCs . Furthermore, foetal MSCs express embryonic pluripotency markers such as Oct-4, Nanog, Rex-1, SSEA-3, SSEA-4, Tra-1–60 and Tra-1–81, again showing their primitiveness . Lastly, apart from the mesodermal lineages, foetal MSCs possess the ability to differentiate into muscle and oligodendrocytes and differentiate more readily into bone, compared with adult MSCs .

Safety of MSCs

MSCs, when grown under normal culture conditions, are commonly considered as safe to transplant, and there are no reports of ectopic tissue formation or malignant transformation . The first clinical transplantation of MSCs was performed over 15 years ago, and adult MSCs have been used in a diverse range of conditions in hundreds of individuals without adverse reactions .

Clinical experience and potential of foetal MSCs

Clinically, allogeneic HLA-mismatched foetal MSCs have been transplanted in utero to two foetuses suffering from severe osteogenesis imperfecta (OI), brittle bone disease, with promising results. In 2002, we performed the first prenatal transplantation of human foetal MSCs in an immunocompetent foetus suffering from OI type III , and, in 2009, a second patient with OI type IV was treated prenatally in collaboration with National University of Singapore and Chang Gung Memorial Hospital in Taiwan .

The transplantation showed promising clinical results with long-term donor-cell engraftment and site-specific differentiation of completely HLA-mismatched MSCs in the bone. The donor-cell engraftment rate varied between 0.003% and 16.6% in the bones. Animal models have shown that despite a low-level engraftment rate of 2%, the donor cells synthesised 20% of the total collagen type I content in the bones , which is also in line with the results from clinical studies of postnatal transplantation for the treatment of OI .

Until 8 years of age, the Swedish patient had only suffered with one fracture and one compression fracture per year. Remarkably, she continued to grow and follow her own height and weight curve at −5 SD (standard deviation), until six years of age, deteriorating to −6.5 SD at 8 years of age. As a result, and due to an increased fracture rate, she was retransplanted with the same-donor MSCs intravenously. Over the following 2 years, she did not acquire any fractures, and her linear growth and mobility improved. We have followed this patient for >13 years, and she is now on a yearly transplantation programme for 4 years with same-donor cells in an attempt to improve her height (years 2013–2016). A child with an identical mutation who did not receive pre- or postnatal MSC transplantation exhibited a very severe phenotype of OI and died as a result of the disease at 5 months of age .

The clinical effect after MSC transplantation is not permanent, and probably regular yearly or biannual infusions are warranted. This has previously been noted in the clinical trials on OI using stem cells postnatally . Although a single prenatal transplantation may not be clinically sufficient for permanent phenotype amelioration, a prenatal transplantation approach is still justifiable as the immunological naïveté of the foetus may ensure the development of immune tolerance towards the donor cells rendering postnatal transplantations more efficient. An early treatment of this severe disorder is also beneficial, both for the child and for the parents.