Fibroblasts and Mesenchymal Stromal/Stem Cells Are Phenotypically Indistinguishable.

BACKGROUND/AIMS: Human mesenchymal stromal/stem cells (MSCs), derived from many different tissues, are characterized by a fibroblast-like morphology, the expression of certain cell surface markers and their ability to differentiate into adipocytes, chondrocytes and osteoblasts. A number of studies have shown that MSCs share many characteristics with fibroblasts; however, there is no well-defined set of phenotypic characteristics that could distinguish between these 2 types of cells. METHODS: We used 4 well-established human fibroblast strains from 3 different tissue sources and several human MSC strains from 2 different tissue sources to compare the phenotypic and immunological characteristics of these cells. RESULTS: Fibroblast strains had a similar morphology to MSCs, expressed the same cell surface markers as MSCs and could also differentiate into adipocytes, chondrocytes and osteoblasts. Also, similar to MSCs, these fibroblasts were capable of suppressing T cell proliferation and modulating the immunophenotype of macrophages. We also show that MSCs deposit extracellular matrices of collagen type I and fibronectin, and express FSP1 in patterns similar to fibroblasts. CONCLUSIONS: Based on currently accepted definitions for cultured human MSCs and fibroblasts, we could not find any immunophenotypic property that could make a characteristic distinction between MSCs and fibroblasts.

Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model.

BACKGROUND AIMS: Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. METHODS: We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4(+) fraction of CD34(+) HSCs, we could improve engraftment. Human cord blood-derived CD34(+) cells and human bone marrow-derived MSCs were used for these studies. RESULTS: When MSCs were transplanted 1 week before CD34(+) cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34(+) cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4(+) cells in the CD34(+) population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. CONCLUSIONS: Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.

Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model.

Until now, ex vivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs in vitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.

In vivo generation of beta-cell-like cells from CD34(+) cells differentiated from human embryonic stem cells.

OBJECTIVE: CD34(+) cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34(+) cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in vivo pancreatic endocrine capacity. MATERIALS AND METHODS: Sheep were transplanted with hESC-derived CD34(+) cells, as well as nonsorted differentiated cultures. Transplantations were carried out with in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult. RESULTS: All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in vivo beta-cell-like activity achieved from the transplantation of the sorted CD34(+) cell population was not augmented by transplanting the entire cell population from which the CD34(+) cells were isolated. Human DNA and insulin messenger RNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from eight animals transplanted with differentiated cell populations and assayed up to 55 months posttransplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell-like activity. Teratomas were absent in the transplanted animals. CONCLUSION: Our data suggest that hESC-derived CD34(+) cells have a potential for long-term in vivo endocrine cellular activity that could prove useful in regenerative medicine. Because the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells.

OBJECTIVE: To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero. MATERIALS AND METHODS: A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow. RESULTS: Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well. CONCLUSION: We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow-derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas.