i. Mesenchymal Stem Cells

A. Immunomodulation by mesenchymal stem cells and clinical experience. Le Blanc K, Ringdén O. J Intern Med. 2007;262:509-525.

This review considers not only the biology of mesenchymal stem cells (MSCs) but also the available results of clinical studies using human MSCs.

The review of the biology of MSCs considers MSC interactions with lymphocytes in vitro; MSC-T cell interaction; MSC effects on APCs, NK cells and B cells; and interactions with lymphocytes in vivo.

Clinical Studies: MSCs have been brought into the clinic for several purposes: to differentiate and heal damaged tissues, to promote hematopoietic engraftment after transplant and for immunosuppression in GVHD.

Metachromatic leukodystrophy and Hurler’s disease: MSCs were expanded in vitro and given IV 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 4 of 5 patients with metachromatic leukodystrophy, there was clear evidence of improvement in nerve conduction velocity.

Osteogenesis imperfecta: Gene-marked MSCs, in order to identify the cells after infusion, were given to 6 children who had undergone HSCT for severe osteogenesis imperfecta. Engraftment of MSCs in the bone and an acceleration of growth velocity were demonstrated. Also, an in utero transplantation was performed using male fetal HLA-mismatched MSCs to a female fetus with severe osteogenesis imperfecta. A bone biopsy showed 0.3-7.4% Y-chromosome-positive cells by FISH analysis, and the patient has had fewer fractures than expected for a child with a severe form of the disease.

HSCT; MSCs were given in a pilot study together with HSCT in 7 patients to enhance engraftment. In 3 of these patients MSCs were given because of previous graft failure and re-transplantation. All patients had 100% donor chimerism within 100 days. This study encourages controlled trials to use MSCs to enhance engraftment after HSCT.

Treatment of GVHD: Haploidentical MSCs were transplanted to a patient with severe treatment-resistant grade IV aGVHD 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. Subsequently 8 patients with steroid-refractory grades III-IV aGVHD and one patient with extensive cGVHD were treated with MSCs. Acute GVHD disappeared completely in 6 of 8 patients and the survival curve was better than that of 16 patients with steroid-resistant biopsy-proven GI GVHD, not treated with MSCs (P=0.003).

Other disorders: Data are also reviewed concerning clinical trials in severe aplastic anemia, severe hemorrhagic cystitis and other instances of severe tissue toxicity.

A table is provided which cites published results of clinical studies in hematological malignancies (n=15), breast cancer (n=28), inborn errors of metabolism (n=11), osteogenesis imperfecta (n= 10), AML (n= 1), leukemia, aplastic anemia, SCID (n= 7), severe aplastic anemia (n=1), hypophosphatasia, Hunter, vasculitis (n=3), leukemia (n=46), malignancies, severe aGVHD (n=9), and tissue toxicity (n= 10).

A total of 117 references are cited.

B. Immunogenicity of umbilical cord tissue derived cells. Cho PS, Messina DJ, Hirsh EL, Chi N, Goldman SN, Lo DP, Harris IR, Popma SH, Sachs DH, Huang CA. Blood. 2008;111:430-438.

Umbilical cord tissue provides a unique source of cells with potential for tissue repair. Umbilical cord tissue-derived cells (UTCs) are MHC class I (MHCI) dull and negative for MHC class II (MHCII), but can be activated to increase MHCI and to express MHCII with IFN-gamma stimulation. Mesenchymal stem cells with similar characteristics have been inferred to be nonimmunogenic; however, in most cases, immunogenicity was not directly assessed.

Using UTC from MHC-defined miniature swine, the authors assessed immunogenicity across a full MHC barrier. Immunogenicity was assessed by in vitro assays including mixed lymphocyte reaction (MLR) and flow cytometry to detect serum alloantibody.

A single injection of MHC-mismatched unactivated UTCs did not induce a detectable immune response. When injected in an inflamed region, injected repeatedly in the same region or stimulated with IFN-gamma prior to injection, UTCs were immunogenic. As clinical cellular repair strategies may involve injection of allogeneic cells into inflamed regions of damaged tissue or repeated doses of cells to achieve the desired benefit, these results on the immunogenicity of these cells in these circumstances may have important implications for optimal success and functional improvement for this cellular treatment strategy for diseased tissues.

C. Comparative generation and characterization of pluripotent unrestricted somatic stem cells with mesenchymal stem cells from human cord blood. Kogler G, Sensken S, Wernet P. Exp Hematol. 2006; 34:1589-95.

Generation and expansion of pluripotent unrestricted somatic stem cells (USSCs) and mesenchymal cells (MSCs) from human cord blood as well their functional characterization were assessed. USSC generation was initiated from fresh and cryopreserved cord blood (CB). Culture conditions and functional assays distinguishing between USSCs and MSCs (e. g., endodermal differentiation) are described.

USSC cultures were initiated from 573 placental CB samples with a total generation frequency of 35.4% (n = 203), whereas successful generation of USSCs from cryopreserved products was scarce. Medium, dexamethasone, and the fetal calf serum source are the major parameters to generate and expand the cells in a pluripotent state (osteogeneic, neural, and endodermal differentiation).

The authors concluded that USSC or MSC cultures can be easily obtained from fresh CB samples and that good manufacturing practice production of such cells is feasible. These USSCs or MSCs from CB, when matched for HLA antigens, may serve as an allogeneic stem cell source for the development of cellular therapy for tissue repair.

D. Co-culture of umbilical cord blood CD34+ cells with human mesenchymal stem cells. Zhang Y, Chai C, Jiang XS, Teoh SH, Leong KW. Tissue Eng. 2006; 12:2161-70.

Insufficient numbers of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) sometimes limit allogenic transplantation of umbilical cord blood (UCB). Ex vivo expansion may overcome this limitation. Mesenchymal stem cells (MSCs), as non-hematopoietic, well-characterized skeletal and connective-tissue progenitor cells within the bone marrow stroma, have been investigated as support cells for the culture of HSCs/HPCs. MSCs are attractive for the rich environmental signals that they provide and for immunological compatibility in transplantation. Thus far, HSC/MSC co-cultures have mainly been performed in 2-dimensional (2D) configuration. We postulate that a 3-dimensional (3D) culture environment that resembles the natural in vivo hematopoietic compartment might be more conducive for regulating HSC expansion. The authors compared the co-culture of HSCs and MSCs in 2D and 3D configurations.

Results demonstrated the benefit of MSC inclusion in HSC expansion ex vivo. Direct contact between MSCs and HSCs in 3D cultures led to statistically significantly higher expansion of cord blood CD34+ cells than in 2D cultures (891- versus 545-fold increase in total cells, 96- versus 48-fold increase of CD34+ cells, and 230- versus 150-fold increase in colony-forming cell assay [CFC]). Engraftment assays in non-obese diabetic/severe combined immunodeficiency mice indicated a high success rate of hematopoiesis reconstruction with these expanded cells.

E. Human mesenchymal stem cells modulate allogeneic immune cell responses. Aggarwal S, Pittenger MF. Blood. 2005;105:1815-22.

The authors point out that mesenchymal stem cells (MSCs) are multipotent cells found in several adult tissues. Transplanted allogeneic MSCs can be detected in recipients at extended time points, indicating a lack of immune recognition and clearance. As well, a role for bone marrow-derived MSCs in reducing the incidence and severity of graft-versus-host disease (GVHD) during allogeneic transplantation has recently been reported; however, the mechanisms remain to be investigated.

The investigators examined the immunomodulatory functions of human MSCs (hMSCs) by coculturing them with purified subpopulations of immune cells and report that hMSCs altered the cytokine secretion profile of dendritic cells (DCs), naive and effector T cells (T helper 1 (T(H)1) and T(H)2), and natural killer (NK) cells to induce a more anti-inflammatory or tolerant phenotype. Specifically, the hMSCs caused mature DCs type 1 (DC1) to decrease tumor necrosis factor alpha (TNF-alpha) secretion and mature DC2 to increase interleukin-10 (IL-10) secretion; hMSCs caused T(H)1 cells to decrease interferon gamma (IFN-gamma) and caused the T(H)2 cells to increase secretion of IL-4; hMSCs caused an increase in the proportion of regulatory T cells (T(Regs)) present; and hMSCs decreased secretion of IFN-gamma from the NK cells. Mechanistically, the hMSCs produced elevated prostaglandin E2 (PGE(2)) in co-cultures, and inhibitors of PGE(2) production mitigated hMSC-mediated immune modulation. The authors suggest that these data offer insight into the interactions between allogeneic MSCs and immune cells and provide mechanisms likely involved with the in vivo MSC-mediated induction of tolerance that could be therapeutic for reduction of GVHD, rejection, and modulation of inflammation.

F. Mesenchymal stem cells: cell biology and potential use in therapy. Kassem M, Kristiansen M, Abdallah BM. Basic Clin Pharmacol Toxicol. 2004;95:209-14.

The authors point out that mesenchymal stem cells are clonogenic, non-hematopoietic stem cells present in the bone marrow and are able to differentiate into multiple mesoderm-type cell lineages e.g. osteoblasts, chondrocytes, endothelial-cells and also non-mesoderm-type lineages e.g. neuronal-like cells. Mesenchymal stem cells with similar biological characteristics to those derived from bone marrow have been isolated from peripheral blood, umbilical cord blood, and other sources. They appear to be ideal candidates for use in regenerative medicine protocols because of their ease of isolation, versatile growth and differentiation potential. Recent studies have demonstrated that the life span of mesenchymal stem cells in vitro can be extended by increasing the levels of telomerase expression in the cells and thus allowing culture of large number of cells needed for therapy. In addition, it has been shown that it is possible to culture the cells in xeno-free environment without affecting their growth or differentiation potential. Finally, the mesenchymal stem cells seem to be hypoimmunogenic and thus allogeneic mesenchymal stem cell transplantation is possible.

G. Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Wang HS, Hung SC, Peng ST, Huang CC, Wei HM, Guo YJ, Fu YS, Lai MC, Chen CC. Stem Cells. 2004;22:1330-7.

Mesenchymal stem cells were originally isolated from bone marrow and recently they have been found to have the potential to differentiate into muscle cells, adipocytes, osteocytes and chondrocytes in culture. The Wharton's jelly of the umbilical cord contains mucoid connective tissue and fibroblast-like cells, and the authors demonstrated that mesenchymal cells isolated from the umbilical cord express matrix receptors (CD44, CD105) and integrin markers (CD29, CD51) but not hematopoietic lineage markers (CD34, CD45). These results suggest that stroma cells from Wharton's jelly are similar to mesenchymal stem cells. Umbilical cord mesenchymal cells can be easily isolated and expanded in culture and induced to differentiate, and this can result in the expression of phenotypes from a variety of lineages. These cells may therefore prove to b a new source of cells for cellular therapies.

H. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE. Stem Cells. 2005;23:220-9.

The authors report a novel harvesting protocol designed to isolate Human Umbilical Cord Perivascular (HUCPV) Cells as a source of mesenchymal progenitors. The method depends on isolation of the umbilical cord vasculature and enzymatic digestion of its perivascular tissue to rapidly harvest a highly proliferative HUCPV population. The authors demonstrated that the resultant cell population possesses a high frequency of colony-forming unit-fibroblast (CFU-F)-deriving cells that proliferate and differentiate rapidly to form bone nodules.

The CFU-F frequency of primary HUCPV cells was 1:333 and the doubling time, which was 60 hours at passage 0 (P0), decreased to 20 hours at P2. This resulted in a significant cell expansion, producing over 10(10) HUCPV cells within 30 days of culture. Furthermore, HUCPV cells cultured in nonosteogenic conditions contained a subpopulation that exhibited a functional osteogenic phenotype and elaborated bone nodules. P0 HUCPV cells contained a 20% subpopulation that presented neither class I nor class II cell-surface major histocompatibility complexes (MHC-/-). This population increased to 95% following passage and cryopreservation. The authors concluded that, due to their rapid doubling time, high frequencies of CFU-F and CFU-O, and high MHC-/- phenotype, HUCPV cells represent a significant source of cells for allogeneic mesenchymal cell-based therapies.

I. Human mesenchymal stem cells support unrelated donor hematopoietic stem cells and suppress T-cell activation. Maitra B, Szekely E, Gjini K, Laughlin MJ, Dennis J, Haynesworth SE, Koc ON. Bone Marrow Transplant. 2004;33:597-604.

Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and represent potential cellular therapy to enhance allogeneic hematopoietic engraftment and prevent graft-versus-host disease (GVHD). In this manuscript the authors showed that coinfusion of human MSCs and unrelated umbilical cord blood (UCB) cells into NOD-SCID mice results in improved frequency and degree of human engfaftment when UCB dose is limiting. When hematopoietic stem cell numbers were limited, human engraftment of NOD-SCID mice was observed only after coinfusion of unrelated human MSCs, but not with coinfusion of mouse mesenchymal cell line. Unrelated human MSCs did not elicit T-cell activation in vitro and suppressed T-cell activation by Tuberculin and unrelated allogeneic lymphocytes in a dose-dependent manner. Cell-free MSC culture supernatant, mouse stromal cells and human dermal fibroblasts did not elicit this effect. The authors indicate that these preclinical data suggest that unrelated, human bone marrow-derived, culture-expanded MSCs may improve the outcome of allogeneic transplantation by promoting hematopoietic engraftment and limiting GVHD and their therapeutic potential should be tested in clinic.

J. Isolation of multipotent mesenchymal stem cells from umbilical cord blood. Lee OK, Kuo TK, Chen WM, Lee KD, Hsieh SL, Chen TH. Blood. 2004;103:1669-75.

Controversy exists as to whether cord blood can serve as a source of mesenchymal stem cells, which can differentiate into cells of different connective tissue lineages such as bone, cartilage, and fat. Little success has been reported in the literature about the isolation of such cells from cord blood. This report describes a novel method to obtain single cell-derived, clonally expanded mesenchymal stem cells that are of multilineage differentiation potential by negative immunoselection and limiting dilution. The immunophenotype of these clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Under appropriate induction conditions, these cells can differentiate into bone, cartilage, and fat. Surprisingly, these cells were also able to differentiate into neuroglial- and hepatocyte-like cells under appropriate induction conditions. Therefore these cells may be more than mesenchymal stem cells as evidenced by their ability to differentiate into cell types of all 3 germ layers.

K. Critical parameters for the isolation of mesenchymal stem cells from umbilical cord blood. Bieback K, Kern S, Kluter H, Eichler H. Stem Cells. 2004;22:625-34.

The authors state that attempts to isolate mesenchymal stem cells (MSC) from umbilical cord blood of full term deliveries have previously either failed or been characterized by a low yield. They investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from umbilical cord blood (UCB) of healthy newborns, and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria.

They were successful in isolating cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) in up to 63% of 59 low-volume UCB units. These were similar to control MSCs from adult bone marrow. The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10⁸ mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition, osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However the MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. The authors concluded that MSC-like cells can be isolated at high efficacy from full-term UCB donations.

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