CD45−/ALDHlow/SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin− Very Small Embryonic-Like (VSEL) Stem Cells Isolated from Umbilical Cord Blood as Potential Long Term Repopulating Hematopoietic Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2444-2444
Author(s):  
Ewa K Zuba-Surma ◽  
Magdalena Kucia ◽  
Rui Liu ◽  
Mariusz Z Ratajczak ◽  
Janina Ratajczak
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

Abstract Recently, we identified a population of very small embryonic-like (VSEL) stem cells in umbilical cord blood (CB) (Leukemia2007;21:297–303) These VSELs are: smaller than erythrocytes; SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−; responsive to SDF-1 gradient; and iv) possessing large nuclei that contain unorganized chromatin (euchromatin). Data obtained in a murine model indicate that a similar cell population isolated from bone marrow (BM) does not reveal hematopoietic activity after isolation. However, in appropriate models (i.e., in vitro co-culture over OP-9 cells or in vivo after intra bone injection), these cells contribute to hematopoiesis and thus possesses potential of long term repopulating hematopoietic stem cells (LT-HSCs). To investigate the hematopoietic activity of CB-derived, CD45 negative VSELs, we employed staining with Aldefluor detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. We sorted CD133+/CD45−/ALDHhigh and CD133+/CD45−/ALDHlow sub-fractions of VSELs from CB samples and established that both freshly sorted CB-derived populations did not grow hematopoietic colonies in vitro. However, when activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and initiate hematopoietic colonies composed of CD45+ cells when replated into methylcellulose cultures. Furthermore, while CD133+/CD45−/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD133+/CD45−/ALDHlow VSELs was delayed. The data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD133+/CD45−/ALDHlow cells (Fig. 1A). In parallel, real time PCR analysis revealed that freshly isolated CD133+/CD45−/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference) while CD133+/CD45−/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference) as compared to total CB mononuclear cells (Fig. B). Furthermore and somewhat unexpectedly, we found that because of their unusually small size, these important cells may be partially depleted (in 42.5±12.6%) during standard preparation strategies of CB units for storage that employ volume reduction. In conclusion, our data suggest very small CB mononuclear cells expressing VSEL markers that are CD133+/CD45−/ALDHlow are highly enriched for the most primitive population of LT-HSCs. These cells may be responsible for long term CB engraftment and be a population of cells from which HSCs should be expanded. We are currently testing this in an in vivo model by performing heterotransplants of CD45− ALDHlow VSELs into immunodeficient mice. It is important to stress that currently employed, routine CB processing strategies may lead up to ~50% loss of these small cells that are endowed with such remarkable hematopoietic activity. Figure Figure

In Vitro and In Vivo Evidence That Umbilical Cord Blood (UCB)-Derived CD45-/SSEA-4+/OCT-4+/CD133+/CXCR4+/Lin- Very Small Embryonic/Epiblast Like Stem Cells (VSELs) Do Not Contain Clonogenic Hematopoietic Progenitors but Are Highly Enriched in More Primitive Stem Cells - Novel View On Hierarchy of UCB Stem Cell Compartment.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 35-35 ◽  
Author(s):  
Ewa K. Zuba-Surma ◽  
Izabela Klich ◽  
Marcin Wysoczynski ◽  
Nicholas J Greco ◽  
Mary J. Laughlin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Stem ◽  
Hematopoietic Activity

Abstract Abstract 35 Recently, we identified in umbilical cord blood (UCB) a population of very small embryonic/epiblast-like (VSEL) stem cells (Leukemia 2007;21:297–303) that are i) smaller than erythrocytes, ii) SSEA-4+/Oct-4+/CD133+/CXCR4+/Lin−/CD45−, iii) respond to SDF-1 gradient and iv) possess large nuclei containing primitive euchromatin. We have demonstrated in vitro that UCB-derived VSELs did not reveal hematopoietic activity freshly after isolation, but grow hematopoietic colonies following co-culture/activation over OP-9 cells. To investigate the hierarchy of UCB-derived, CD45 negative VSELs, we employed staining with Aldefluor - detecting aldehyde dehydrogenase (ALDH), the enzyme expressed in primitive hematopoietic cells. Subsequently, we sorted CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow sub-fractions of VSELs from UCB samples and established that freshly sorted from UCB VSELs in contrast to sorted CD45+/ CD133+/ALDHhigh and CD45+/CD133+/ALDHlow hematopoietic stem cells (HSC) did not grow colonies in vitro. However, when CD45− VSELs were activated/expanded over OP-9 stroma cells, they exhibit hematopoietic potential and grew in routine methylcellulose cultures hematopoietic colonies composed of CD45+ cells. Interestingly, while CD45−/CD133+/ALDHhigh VSELs gave raise to hematopoietic colonies after the first replating, the formation of colonies by CD45−/CD133+/ALDHlow VSELs was somehow delayed, what suggest that they needed more time to acquire hematopoietic commitment. Thus our in vitro data indicate that both populations of CD45− cells may acquire hematopoietic potential; however hematopoietic specification is delayed for CD45−/CD133+/ALDHlow cells, suggesting their more primitive nature. In parallel, real time PCR analysis confirmed that while freshly isolated CD45−/CD133+/ALDHhigh VSELs express more hematopoietic transcripts (e.g., c-myb, 80.2±27.4 fold difference), CD45−/CD133+/ALDHlow exhibit higher levels of pluripotent stem cell markers (e.g., Oct-4, 119.5±15.5 fold difference as compared to total UCB mononuclear cells) (Figure 1 panel A). Next hematopoietic potential of UCB-derived VSELs was tested in vivo after transplantation into NOD/SCID mice (Figure 1 panel B and C). We noticed that both CD45−/CD133+/ALDHhigh and CD45−/CD133+/ALDHlow VSELs, give rise to human lympho-hematopoietic chimerism in lethally irradiated NOD/SCID mice as assayed 4–6 weeks after transplantation. The level of human hematopoietic CD45+ cells in murine peripheral blood (PB), bone marrow (BM) and spleen (SP) were comparable for both transplanted UCB-VSELs fractions - 7.1±2.9% (PB), 23.2±0.2% (SP) and 25.2±1.0% (BM). In conclusion, our data suggest that freshly isolated very small CD45 negative UCB-VSELs are depleted from clonogeneic progenitors, however they are highly enriched for primitive HSC. Based on our in vitro and in vivo data we postulate following hierarchy of hematopoietic stem cells in UCB (from most primitive to more differentiated) i) CD45−/CD133+/ALDHlow, ii) CD45−/CD133+/ALDHhigh , iii) CD45+/CD133+/ALDHlow and iv) CD45−/CD133+/ALDHhigh. We also postulate that as we have already shown for murine BM-derived VSELs, human UCB-derived CD45 negative VSELs correspond to a population of most primitive long term repopulating HSC (LT-HSC). Of note, we also found that currently employed, routine UCB processing strategies may lead up to ∼50% unwanted loss of these small cells that are endowed with such remarkable hematopoietic activity! Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematopoietic stem/progenitor cells, generation of induced pluripotent stem cells, and isolation of endothelial progenitors from 21- to 23.5-year cryopreserved cord blood

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4773-4777 ◽  
Author(s):  
Hal E. Broxmeyer ◽  
Man-Ryul Lee ◽  
Giao Hangoc ◽  
Scott Cooper ◽  
Nutan Prasain ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Induced Pluripotent

Abstract Cryopreservation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) is crucial for cord blood (CB) banking and transplantation. We evaluated recovery of functional HPC cryopreserved as mononuclear or unseparated cells for up to 23.5 years compared with prefreeze values of the same CB units. Highly efficient recovery (80%-100%) was apparent for granulocyte-macrophage and multipotential hematopoietic progenitors, although some collections had reproducible low recovery. Proliferative potential, response to multiple cytokines, and replating of HPC colonies was extensive. CD34+ cells isolated from CB cryopreserved for up to 21 years had long-term (≥ 6 month) engrafting capability in primary and secondary immunodeficient mice reflecting recovery of long-term repopulating, self-renewing HSCs. We recovered functionally responsive CD4+ and CD8+ T lymphocytes, generated induced pluripotent stem (iPS) cells with differentiation representing all 3 germ cell lineages in vitro and in vivo, and detected high proliferative endothelial colony forming cells, results of relevance to CB biology and banking.

scholarly journals Short- and Long-Term Multilineage Repopulating Hematopoietic Stem Cells in Late Fetal and Newborn Mice: Models for Human Umbilical Cord Blood

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 174-181 ◽  
Author(s):  
David E. Harrison ◽  
Clinton M. Astle
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord Blood ◽  
Functional Abilities ◽  
Newborn Mice ◽  
Hematopoietic Stem ◽  
Short And Long Term ◽  
Marrow Cells

Abstract Blood from late fetal and newborn mice is similar to umbilical cord blood obtained at birth in human beings, an important source of stem cells for clinical transplantation. The mouse model is useful because long-term functions can be readily assayed in vivo. To evaluate the functions of hematopoietic precursors in the blood and other tissues of late fetal and newborn mice, short- and long-term multilineage repopulating abilities were measured in vivo by competitive repopulation. Manipulations that might affect cell function, such as enrichment, tissue culture, or retroviral marking, were avoided. Hematopoietic stem cell functions of late fetal or newborn blood, liver, and spleen, were assayed as myeloid and lymphoid repopulating abilities relative to standard adult marrow cells. Donor cells from these tissues as well as adult control donor marrow cells were all of the same genotype. Cells from each donor tissue were mixed with portions from a pool of standard adult “competitor” marrow distinguished from the donors by genetic differences in hemoglobin and glucosephosphate isomerase. After 21 to 413 days, percentages of donor type myeloid and lymphoid cells in recipient blood were measured to assay the functional abilities of donor precursors relative to the standard. These relative measures are expressed as repopulating units, where each unit is equivalent to the repopulating ability found in 100,000 standard adult marrow cells. Thus, measures of repopulating units do not compare single cells but overall repopulating abilities of donor cell populations. Relative functional abilities in 1 million nucleated cells from late fetal or newborn blood were several times less than those found in adult marrow, but far more than in normal adult blood, and appeared to include long-term functional primitive hematopoietic stem cells (PHSC) similar to those in marrow. To estimate functional abilities of individual PHSC, variances among large groups of identical recipients were analyzed using both the binomial model and competitive dilution, a new model based on the Poisson distribution. The data best fit the hypothesis that individual PHSC from adult marrow, late fetal blood, or newborn blood each produce similar fractions of the total lymphoid and erythroid cells found in the recipient for many months.

scholarly journals Influence of the endothelial cells on the erythroid differentiation of umbilical cord blood hematopoietic stem cells in vitro

2020 ◽  
Vol 17 (1) ◽  
pp. 78-86
Author(s):  
A. S. Voytehovich ◽  
E. V. Vasina ◽  
V. S. Kastsiunina ◽  
I. N. Seviaryn ◽  
N. V. Petyovka
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Vascular Niche

The objective is to study the effect of umbilical cord blood endothelial cells on the hematopoietic cells growth and the maturation in the erythroid direction in co-culture, as well as the expression of adult and fetal hemoglobin genes during erythroid differentiation under the conditions of vascular niche modeling in vitro. We used the following research methods: cultural, flow cytometry, real-time PCR and morphological analysis. We have developed the method of hematopoietic cord blood stem cells erythroid differentiation in co-culture using cord blood endothelial cell progenitors. CD34+CD31+CD144+CD105+CD90–CD45– progenitors of endothelial cells stimulate the erythroid differentiation of hematopoietic CD34+ cord blood cells and the growth of erythroid progenitors in co-culture from the 4th to 11th day in the presence of the stem cell factor, the erythropoietin and the fibroblast growth factor-2. The in vitro modeling of the vascular niche increases the mature CD36–CD235a+ erythroid cells 2.5 times higher than those in the liquid culture. The microenvironment of endothelial cells does not affect the level and expression ratio of fetal and adult hemoglobin during the erythroid differentiation in vitro.

The Feature of SALL4 and BMI-1 Expression in Placenta and Umbilical Cord Blood

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4800-4800
Author(s):  
Shaohua Chen ◽  
Qi Shen ◽  
Lijian Yang ◽  
Bo Li ◽  
Yupo Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Research Funding ◽  
Mononuclear Cells ◽  
Expression Level ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Expression Levels

Abstract Abstract 4800 The SALL4 is a newly discovered pluripotency stem cell factor involved in the self-renewing, hematopoietic stem cells (HSCs) and leukemic stem cells. Its important role in normal HSCs and leukemic stem cells is supported by its interactions with several key players, like BMI-1, a putative oncogene of the Polycomb group family, controlling specific target genes involved in development, cell differentiation, proliferation, and senescence. Little is known the expression feature of SALL4 and BMI-1 mRNA in placenta and umbilical cord blood (UCB). In this study, the mRNA expression level of SALL4 and BMI-1 in 10 cases placenta tissues and the mononuclear cells from the corresponding UCB (CBMCs) were measured by real-time PCR with SYBR Green I technique, β2-microglobulin gene (β2M) was used as an endogenous reference, mononuclear cells from 10 healthy adults peripheral blood (PBMCs) and 2 cases of adult thymic cells served as control. The result showed that the expression level of both SALL4 and BMI-1 in placenta (Median: 34.36 and 17.55 respectively) were significantly higher than those from CBMCs (Median: 11.3 and 2.07 respectively) (p=0.007, p=0.001) and PBMCs (Median: 0.64 and 0.03 respectively) (P<0.001, P<0.001). Moreover, both SALL4 and BMI-1 expression levels in CBMCs were significant higher than those from PBMCs (P=0.002, P=0.001). And the expression of SALL4 could be detected in thymic cells. Correlation analysis was performed among the relative expression levels of SALL4 and BMI-1. The results showed that significant positive correlation between the expression levels of SALL4 and BMI-1 was observed in placenta tissues and CBMCs, as well as in PBMCs (rs=0.648, P=0.043, rs=0.721, P=0.019, rs=-0.697, P=0.025). In conclusions, the expression level of SALL4 and BMI-1 might be related to the numbers of hematopoietic stem cells, placenta tissues may be as an appropriate model for investigating gene regulation of SALL4 and BMI-1, while the significance of expression both genes in PBMCs and thymic cells is needed further investigation. Disclosures: Chen: National Natural Science Foundation of China(no. 30871091): Research Funding; the Fundamental Research Funds for the Central Universities (No. 21610603): Research Funding.

Human CD34+ Haematopoietic Stem Cells (HSC) from Umbilical Cord Blood Display an Endodermal Phenotype When Exposed to Activin A In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1697-1697
Author(s):  
Claudia Albera ◽  
Anne E. Bishop ◽  
Julia M. Polak ◽  
Nicki Panoskaltsis
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Activin A ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Transcript Levels ◽  
Time Points

Abstract Adult stem cells may not be tissue specific and transdifferentiation into cells committed to other tissues may occur. In vivo, bone marrow HSC can contribute to cells characteristic of resident endoderm-derived tissues, such as lung. We hypothesized that if transdifferentiation can occur, then in vitro evidence could be obtained from an enriched population of hematopoietic cells. This study evaluated whether endodermal lineages could be derived from human umbilical cord blood (UCB) cells in order to provide a novel source for the regeneration of non-hematopoietic tissues, such as the lung. UCB CD34+ hematopoietic stem cells were magnetically enriched to a purity greater than 92%. A fraction of these was immediately characterised by flow cytometry and the remaining cells were expanded for 3 days in serum-free media. The effects of a combination of growth factors on these cells, including FGF-4, HGF and Activin A, were evaluated after 15 and 30 days of culture. RT-PCR analysis revealed detectable mRNA levels from representative endodermal genes (SOX17, GATA4 and FOXa2) after 15 days of induction and epithelial genes cytokeratin (CK)-18 and E-cadherin (E-cad) after 30 days of induction. Control cells after 3 days expansion and freshly isolated CD34+ (day 0) showed detectable transcript levels of HOXb4, Gata1 and PECAM-1 but they were negative for all endoderm markers. A panel of endodermal and epithelial primary antibodies, GATA4, GATA6, E-cad, CK-18, Pan-CK and Albumin were immunolocalized in cultures after 15 and 30 days of induction showing gene and protein expression. In order to evaluate the temporal expression pattern of the endodermal genes excluding the endothelial cell component, enriched CD34+ cells were subsequently FACS-sorted to yield a CD34+CD45+ cell population of greater than 98% purity. Quantitative real time RT-PCR was used to measure fold changes in the expression of GATA4, FOXa2, SOX17, E-cad and CK-18 genes in response to low and high Activin A concentrations at 6 different time points of induction cell culture (0, 3, 5, 7, 9, 12 and 15 days). Exposure to Activin A increased the expression of the endoderm transcript levels as early as 3 days of induction for E-cad and SOX17 and at 7 and 9 days of induction for GATA4 and FOXa2, respectively. Data were normalized to the housekeeping gene ß-actin and were expressed relative to the level of the gene of interest at time zero. HSC cultivated without Activin A and evaluated at the same time points did not express endoderm transcripts. These findings show that in the presence of Activin A, hematopoietic stem cells can be induced to express genes and proteins indicative of endoderm lineage in vitro. Since Activin A is upregulated in damaged tissue and is required for wound healing, the data suggest a mechanism for in vitro “transdifferentiation” of HSC, especially in the repair of damaged organs, and indicate that this process may be applied therapeutically for regenerative medicine.

Expansion of Umbilical Cord Blood Hematopoietic Stem Cells for Clinical Use.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4049-4049
Author(s):  
Rowayda Peters ◽  
Gregor Hutter ◽  
Adriano Aguzzi ◽  
Mathias Heikenwaelder ◽  
Christoph Renner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Cell Count ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Induction Medium ◽  
Adherent Cells ◽  
Hematopoietic Stem

Abstract Umbilical cord blood (UCB) is an alternative donor source for allogeneic hematopoietic stem cell transplantation. However, transplantation in adults is frequently limited by the small number of cells available in a unit. We have previously developed the technology to expand hematopoietic stem cells (HSC) and stromal/mesenchymal stem cells (SMSC) from all UCB frozen samples (13, 3). The incubation of thawed UCB mononuclear cells (MNC) in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB HSC (non-adherent). Also, long-term expansion of SMSC (adherent cells) was successful. In order to upscale the expansion to use for clinical applications, we analyzed 3 frozen UCB comparing the expansion from 24-well plates (previous) versus expansion in bags (VueLife™) after 10 and 14 days of culture. Results show substantial expansion in total cell count (TCC, 4.8, 10.9: 2.4, 3.8 fold) at d10 and d14 from wells & bags respectively. TCC increased further in the presence of SMSC (38% & 33% in CD34+ cell count cultured in wells). CD133+CD34+CD38- HSC multiplied (11, 25 fold, d10/d14 & colony forming cells (CFC) 19 fold at d14 bags respectively). Heterogeneous cell populations were detected after d 14 in bags: T and B -lymphoid (%CD3/%CD19; 65/4:50w/3), megakaryocytic (%CD61; 7:4) and myeloid (%CD33; 31:43) at d0/d14 respectively. Further, expanded cells (250,000) containing a small number of CD133+CD34+CD38- (15,000–30,000) were injected into the liver of sub-lethally irradiated newborn Balb/C Rag2-Cγ−/− mice. Our preliminary data show no engraftment from cells expanded in wells and bags after 6 weeks of transplantation from d10 cultures (human CD45 + <1%). However, positive engraftment in mouse PB was detected from cells expanded for 14 d (wells, 1.16–2.5% & bags, 1.21–3.9%) as compared to control mice (CD45; 30% PB & 70% BM) receiving selected CD34 + (300,000 CD34+ at d0). Primitive repopulating cells (PRC), and multilineage human CFC were detected after transplantation. On d14 of HSC expansion, UCB MNC derived adherent cells (SMSC) were enriched by trypsinization. SMSC were established in serum free and serum plus culture as well. The immunophenotype of harvested SMSC was CD29+, CD44+ and CD45−, CD34− and CD133− at percentages + >90%. Following repeated trypsinization, SMSC count increased 41–96 folds. CFU-Fibroblast colonies (92–173) were generated from 104 SMSC after 2 weeks in MesenCult medium. We have previously differentiated SMSC into hepatocytes. Now we also generated adipocytes in an induction medium containing, Insulin, dexamethasone & indomethacin. SMSC formed Oil-droplet vacuoles in the cytoplasm in 3 weeks. The culture conditions we defined to maintain UCB PRC should be developed clinically. SMSC described herein exhibit in vitro properties of multipotent stem cells.

Novel Evidence for the Presence of Potent, Paracrine, Pro-Angiopoietic Effects of Purified Human Umbilical Cord Blood-Derived CD133+ Cells - Implications for Adult Stem Cell Therapies in Regenerative Medicine

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4740-4740
Author(s):  
Kasia Mierzejewska ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Adult Stem Cell ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Cell Therapies

Abstract Abstract 4740 Background. As populations of CD34+, CD34+CXCR4+, or CD133+ cells that are enriched in stem cells, adult stem and progenitor cells purified from bone marrow (BM), mobilized peripheral blood (mPB), and umbilical cord blood (UCB) are currently employed in the clinic to treat damaged organs (e.g., heart after myocardial infarction [AMI] or injured spinal cord or liver). The cell populations expressing these phenotypes are highly enriched primarily for hematopoietic stem/progenitor cells (HSPCs) and small numbers of endothelial progenitors, and for many years it has been wrongly supposed that they can trans-dedifferentiate into tissue-specific cells. However, even when improvement of organ function is observed after employing them in therapy, the lack of a convincing demonstration for the presence of donor-recipient chimerism in treated tissues in most of the studies performed so far indicates that mechanisms other than trans-dedifferentiation of the HSPCs delivered to the damaged organs into tissue-specific cells play a significant role in some positive clinical outcomes. In support of this conclusion, evidence has accumulated that stem cells secrete a variety of growth factors, cytokines, chemokines, and bioactive lipids that interact with the surrounding microenvironment and, when used in therapy, improve cell viability in damaged organs. In particular, more attention is currently being paid to microvesicles (MVs), which are shed from the cell surface or derived from the intracellular membrane compartment as mediators in cell-to-cell communication. Hypothesis. We hypothesized that these positive outcomes in adult stem cell therapies (e.g., by employing CD133+ cells) can be explained by the paracrine effects of these cells, involving both soluble factors as well as cell membrane-derived MVs. Experimental strategies. CD133+ cells were purified from UCB by employing immunomagnetic beads (> 95% purity as checked by FACS) and incubated for 24 hours in RPMI at 37°C in a small volume of medium supplemented with 0.5% albumin. Subsequently, we harvested conditioned media (CM) from these cells and isolated CD133+ cell-shed microvesicles (MVs) by high speed centrifugation. We employed sensitive ELISA assays to measure the concentration of important pro-angiopoietic and anti-apoptotic factors in CD133+ cell-derived CM and isolated mRNA from both CD133+ cells and CD133+ cell-derived MVs for RQ-PCR analysis of gene expression. Subsequently, the chemotactic activity of CD133+ cell-derived CM and MVs was tested against human umbilical cord blood endothelial cells (HUVECs), and, in parallel, we tested whether CD133+ cell-derived CM and MVs induce major signaling pathways in HUVECs. Finally, in in vitro functional assays, we tested the ability of CD133+ cell-derived CM and MVs to induce tube formation by HUVECs and the ability of in vivo Matrigel assay implants to induce angiogenesis. Results. We observed that highly purified UCB-derived CD133+ cells express mRNAs and secrete proteins for several pro-angiopoietic factors (e.g. VEGF, KL, FGF-2, and IGF-1) into CM and shed microvesicles (MVs) from the cell surface and endosomal compartment that are enriched for mRNAs encoding VEGF, KL, FGF-2, and IGF-1. Both CD133+ cell-derived CM and MVs possessed anti-apoptotic properties, increased the in vitro cell survival of endothelial cells, stimulated phosphorylation of MAPKp42/44 and AKT in HUVECs, induced chemotactic migration, proliferation and tube formation in vitro in HUVECs, as well as stimulated in vivo angiogenesis in Matrigel implants. Conclusions. These observations suggesting an important role for CD133+ cell-derived paracrine signals should be considered when evaluating clinical outcomes using purified CD133+ cells in regenerative medicine. Overall, these cell-derived paracrine signals may explain the therapeutic benefits of adult stem cells employed in regeneration of, for example, heart AMI. Finally, we will discuss several possibilities for enhancing secretion and modulating the composition of these paracrine signals that could be explored in the clinic. Disclosures: No relevant conflicts of interest to declare.

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