Long-Term Effects of Bone Marrow-Derived Mesenchymal Stem Cells in Dextran Sulfate Sodium-Induced Murine Chronic Colitis

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

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Perivascular/Mesenchymal Stem Cells from Multiple Human Tissues Support Hematopoiesis in Vitro.

Blood ◽

10.1182/blood.v112.11.1361.1361 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1361-1361

Author(s):

Elisa Montelatici ◽

Gabriella Andriolo ◽

Mihaela Crisan ◽

Rosaria Giordano ◽

Paolo Rebulla ◽

...

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Stromal Cells ◽

Marrow Stromal Cells ◽

Hematopoietic Cell ◽

Human Tissues

Abstract Mesenchymal stem cells (MSC) can be derived selectively in culture from multiple organs, an omnipresence we have recently suggested to be explained by the perivascular location of native MSC ancestors within intact tissues (Crisan et al. 2008, in press). We have now analyzed the ability of MSC extracted pro- or retrospectively from different human tissues to support hematopoiesis. MSC were either classically derived in primary cultures of umbilical cord blood (UCB) lineage-depleted mononuclear cells (n=3) or enzymatically dissociated adult adipose tissue (n=3), or grown as CD146+ NG2+ CD34-CD56- CD45- pericytes (n=2) purified by flow cytometry from fetal skeletal muscle and cultured over the long term. In both settings, identical MSC were obtained that maintained a stable CD146+ CD90+ CD73+ CD105+ CD34- CD45- surface phenotype and could differentiate into skeletal muscle, fat, bone and cartilage. CD34+ hematopoietic progenitors (n=3) immunoselected from term UCB were seeded (5×10e3cells/cm2 in triplicate) onto confluent irradiated layers of MSC derived from UCB, adipose tissue or fetal muscle pericytes (MSCu, MSCa and MSCmp, respectively) or, as a control, MS5 bone marrow stromal cells that allow the proliferation of very primitive human progenitor cells. All studies were approved by the relevant institutional regulatory board. The cells were cocultured for 5 weeks in a classical long-term culture-initiating cell assay in a complete medium (MyeloCult H5100, Stem Cell Technologies) containing hydrocortisone but no added cytokine. Wells were scored daily for the presence of cobblestone areas (CA) and half of the medium was replaced every week. Eventually, trypsinized cells from each well were characterized by flow cytometry for the expression of hematopoietic cell markers and assayed for CFC potential. After 14 days of incubation, colonies grown in semi-solid medium were scored as derived from colony forming units (CFU)-granulocyte, erythroid, macrophage, megakaryocyte (GEMM) and as high-proliferative-potential colony precursors (HPPC), the most primitive hematopoietic cell so far identified in a clonogenic assay in vitro. Within the CD45+ gate, all trypsinized cultures contained comparable percentages of CD34+lin- cells (MSCu: 51±9%; MSCa: 58±14%; MSCmp: 61±19%; MS5: 59±18%), the most immature hematopoietic cell compartment maintained during the long-term coculture. MSCu and MSCmp supported a similar cell proliferation during the whole culture while on MSCa, CA formed very rapidly and consistently but eventually decreased over the long-term culture. Interestingly, MSCu and MSCmp supported the development of the highest numbers of HPPC and of CFU giving rise to the largest GEMM colonies, as compared to MSCa that gave the same results as the control MS5 cell line. In summary, all MSCs tested were able to support hematopoiesis and CA formation, albeit with differences in growth kinetics and morphology of the colonies. Herein we show for the first time that purified human perivascular cells exhibit robust hematopoiesis support in vitro, in addition to multilineage mesodermal developmental potential. In conclusion, we demonstrate that MSC from novel sources distinct from the bone marrow are able to support hematopoiesis. These results further sustain the identity, beyond acronyms, between marrow stromal cells, long known for their support of hematopoiesis, and mesenchymal stem cells that gained more recent credit in the field of regenerative medicine because of their multilineage differentiation potential.

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