Abhilasha Tiwari

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line.

Lineage‐specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell‐free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell‐free matrices from a murine bone marrow stromal cell line (MS‐5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellular matrices were used as biological scaffolds for the lineage‐specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony‐forming assays. We could get up to 80‐fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering‐based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow. Copyright

Osteohematopoietic stem cell niches in bone marrow.

In adult mammals, maturation of blood and bone cells from their respective progenitors occurs in the bone marrow. The marrow region contains many progenitor and stem cell types that are confined by their biochemical and cellular microenvironments, referred to as stem cell niches. The unique properties of each niche assist the survival, proliferation, migration, and differentiation of that particular stem or progenitor cell type. Among the different niches of the bone marrow, our understanding of the osteohematopoietic niche is the most complete. Its properties, described in this chapter, are a model for studying adult stem cell differentiation, but a lot remains unknown. Our improved understanding of hematopoietic stem cell biology and its relationship with the properties of these niches are critical in the effective and safe use of these cells in regenerative medicine. Here, we review the current knowledge on the properties of these niches and suggest how the potential of hematopoietic progenitors can be utilized in regenerative medicine.

Expansion of Human Hematopoietic Stem/Progenitor Cells on Decellularized Matrix Scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic human-body-niche-like conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.

The mouse endometrium contains epithelial, endothelial and leucocyte populations expressing the stem cell marker telomerase reverse transcriptase.

STUDY HYPOTHESISnThe mouse endometrium harbours stem/progenitor cells that express the stem cell marker mouse telomerase reverse transcriptase (mTert).nnnSTUDY FINDINGnWe used a mouse carrying a transgenic reporter for mTert promoter activity to identify rare endometrial populations of epithelial and endothelial cells that express mTert.nnnWHAT IS KNOWN ALREADYnStem/progenitor cells are hypothesized to be responsible for the remarkable regenerative capacity of the endometrium, but the lack of convenient endometrial stem/progenitor markers in the mouse has hampered investigations into the identity of these cells.nnnSTUDY DESIGN, SAMPLES/MATERIALS, METHODSnA mouse containing a green fluorescent protein (GFP) reporter under the control of the telomerase reverse transcriptase promoter (mTert-GFP) was used to identify potential stem/progenitor cells in the endometrium. mTert promoter activity was determined using fluorescence microscopy and flow cytometry to identify GFP(+) cells. GFP(+) cells were examined for epithelial, stromal, endothelial, leucocyte and proliferation markers and bromodeoxyuridine retention to determine their identity. The endometrium of ovariectomized mice was compared to that of intact cycling mice to establish the role of ovarian hormones in maintaining mTert-expressing cells.nnnMAIN RESULTS AND THE ROLE OF CHANCEnWe found that mTert-GFP is expressed by rare luminal and glandular epithelial cells (0.3% of epithelial cells by flow cytometry), rare CD45(-) cells in the stromal compartment (0.028 ± 0.010% of stromal cells by microscopy) and many CD45(+) leucocytes. Ovariectomy resulted in significant decrease of mTert-GFP(+) epithelial cells (P = 0.029 for luminal epithelium; P = 0.034 for glandular epithelium) and a decrease in the percentage of mTert-GFP(+) CD45(+) leucocytes in the stromal compartment (P = 0.015). However, CD45(-) mTert-GFP(+) cells in the stromal compartment were maintained in ovariectomized mice. This population is enriched for cells bearing the endothelial marker CD31 (10.3% of CD90(-) CD45(-) and 97.8% CD90(+) CD45(-) by flow cytometry). CD45(-) mTert-GFP(+) cells also immunostained for the endothelial marker von Willebrand factor. These results suggest that the endometrial epithelium and vasculature are foci of stem/progenitor activity and provide a system to investigate molecular mechanisms involved in endometrial regeneration and repair.nnnLIMITATIONS, REASONS FOR CAUTIONnThe stem/progenitor activity of endometrial mTert-GFP(+) cells needs to be experimentally verified.nnnWIDER IMPLICATIONS OF THE FINDINGSnThe identification and characterization of mTert-expressing progenitor cells in the mouse will facilitate the identification of equivalent populations in the human endometrium that are likely to be involved in endometrial function, fertility and disease.nnnLARGE-SCALE DATAnNot applicable.nnnSTUDY FUNDING AND COMPETING INTERESTSnThis study was funded by National Health and Medical Research Council (NHMRC) of Australia grants (1085435, C.E.G., J.A.D.), 1021127 (C.E.G.), NHMRC Senior Research Fellowship (1042298, C.E.G.), the Victorian Infrastructure Support Program, U.S. National Institutes of Health grant R01 DK084056 (D.T.B.) and the Harvard Stem Cell Institute (D.T.B.). The authors have no conflicts of interest to declare.

Comparative Gene Expression Profiling of Stromal Cell Matrices that Support Expansion of Hematopoietic Stem/Progenitor Cells

The bone marrow microenvironment maintains a stable balance between self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs). This microenvironment, also termed the “hematopoietic niche”, is primarily composed of stromal cells and their extracellular matrices (ECM) that jointly regulate HSPC functions. Previously, we have demonstrated that umbilical cord blood derived HSPCs can be maintained and expanded on stromal cell derived cellular matrices that mimic the complexity of the hematopoietic niche. The results indicated that matrices prepared at 20% O2 with osteogenic medium (OGM) were best suited for expanding committed HSPCs, whereas, matrices prepared at 5% O2 without OGM were better for primitive progenitors. Based upon these results we proposed that individual constituents of these matrices could be responsible for regulation of specific HSPC functions. To explore this hypothesis, we have performed comparative transcriptome profiling of these matrix producing cells, which identified differential expression of both known niche regulators, such as Wnt4, Angpt2, Vcam and Cxcl12, as well as genes not previously associated with HSPC regulation, such as Depp. MetaCore analysis of the differentially expressed genes suggests the down-regulation of several ECM related pathways and up-regulation of Ang-Tie2 and Wnt signaling pathways in OGM under high O2 (20%). Our findings provide an overview of several known and unique genes and pathways that play potential key roles in the support of HSPCs by stromal cells, both ex vivo and in vivo, and could be helpful in understanding the complex network of signaling and communication in hematopoietic niches.

UNIT 1C.15 Expansion of Human Hematopoietic Stem/Progenitor Cells on Decellularized Matrix Scaffolds

Umbilical cord blood (UCB) is one of the richest sources for hematopoietic stem/progenitor cells (HSPCs), with more than 3000 transplantations performed each year for the treatment of leukemia and other bone marrow, immunological, and hereditary diseases. However, transplantation of single cord blood units is mostly restricted to children, due to the limited number of HSPC per unit. This unit develops a method to increase the number of HSPCs in laboratory conditions by using cell-free matrices from bone marrow cells that mimic human-body niche-like conditions as biological scaffolds to support the ex vivo expansion of HSPCs. In this unit, we describe protocols for the isolation and characterization of HSPCs from UCB and their serum-free expansion on decellularized matrices. This method may also help to provide understanding of the biochemical organization of hematopoietic niches and lead to suggestions regarding the design of tissue engineering-based biomimetic scaffolds for HSPC expansion for clinical applications.

Preterm umbilical cord blood derived mesenchymal stem/stromal cells protect preterm white matter brain development against hypoxia-ischemia

Introduction Preterm infants are at high risk for white matter injury and subsequent neurodevelopmental impairments. Mesenchymal stem/stromal cells (MSC) have anti‐inflammatory/immunomodulatory actions and are of interest for neural repair in adults and newborns. This study examined the neuroprotective effects of allogeneic MSC, derived from preterm umbilical cord blood (UCB), in a preterm sheep model of white matter injury. Methods Quad‐lineage differentiation, clonogenicity and self‐renewal ability of UCB‐derived MSC were confirmed. Chronically instrumented fetal sheep (0.7 gestation) received either 25 min hypoxia‐ischemia (HI) to induce preterm brain injury, or sham‐HI. Ten million MSC, or saline, were administered iv to fetuses at 12 h after HI. Fetal brains were collected 10d after HI for histopathology and immunocytochemistry. Results HI induced white matter injury, as indicated by a reduction in CNPase‐positive myelin fiber density. HI also induced microglial activation (Iba‐1) in the periventricular white matter and internal capsule (P < .05 vs control). MSC administration following HI preserved myelination (P < .05), modified microglial activation, and promoted macrophage migration (CD163) and cell proliferation (Ki‐67) within cerebral white matter (P < .05). Cerebral CXCL10 concentration was increased following MSC administration (P < .05), which was likely associated with macrophage migration and cell proliferation within the preterm brain. Additionally, MSC administration reduced systemic pro‐inflammatory cytokine TNF&agr; at 3d post‐HI (P < .05). Conclusions UCB‐derived MSC therapy preserved white matter brain structure following preterm HI, mediated by a suppression of microglial activation, promotion of macrophage migration and acceleration of self‐repair within the preterm brain. UCB‐derived MSC are neuroprotective, acting via peripheral and cerebral anti‐inflammatory and immunomodulatory mechanisms. HighlightsPreterm UCB‐MSC infusion reduces HI‐induced white matter injury in the preterm brainMSC induce macrophage migration and oligodendrocyte proliferation within white matterMSC suppress systemic TNF‐a release and cerebral microglial activation following HI

Controlling the Effective Oxygen Tension Experienced by Cells Using a Dynamic Culture Technique for Hematopoietic Ex Vivo Expansion

Clinical hematopoietic stem/progenitor cell (HSPC) transplantation outcomes are strongly correlated with the number of cells infused. Hence, to generate sufficient HSPCs for transplantation, the best culture parameters for expansion are critical. It is generally assumed that the defined oxygen (O2 ) set for the incubator reflects the pericellular O2 to which cells are being exposed. Studies have shown that low O2 tension maintains an undifferentiated state, but the expansion rate may be constrained because of limited diffusion in a static culture system. A combination of low ambient O2 and dynamic culture conditions has been developed to increase the reconstituting capacity of human HSPCs. In this unit, the protocols for serum-free expansion of HSPCs at 5% and 20% O2 in static and dynamic nutrient flow mode are described. Finally, the impact of O2 tension on HSPC expansion in vitro by flow cytometry and colony forming assays and in vivo through engraftment using a murine model is assessed.