Distinct Differentiation Potential Research Articles

Molecular-genetic and immunophenotypic analysis of antigen profile and osteogenic and adipogenic potentials of mesenchymal stromal cells from fetal liver and adult bone marrow in rats

Comparative characteristics of mesenchymal stromal cells (MSC) from fetal liver and adult bone marrow are of great interest due to similar contribution to organization of the hematopoietic microenvironments in these organs in different developmental periods. It is known that MSC play a pivotal role in organization of hematopoietic stem cells "niches". Moreover, the histogenetic affinity between MSC from both sources is not ruled out. Antigen profiling using immunocytochemistry and RT-PCR confirmed that cell populations under study corresponded to MSC criteria and had no hematopoietic, lymphoid and endothelial cells after second passage. Comparative analysis of osteogenic and adipogenic marker expression revealed distinct differentiation potentials of MSC from two hematopoietic organs: adipogenic potentials of hepatic MSC was weaker but ability to differentiate to osteogenic lineage was extremely low, if any, in comparison with marked osteo- and adipogenic potentials of adult bone marrow MSC. Similar cell phenotype but unequal differentiation potentials under identical conditions in vitro may be attributed to a distinct developmental program in pre- and postnatal histogenesis of MSC from different sources.

The Side Population Contains Functionally Distinct Hematopoietic Stem Cell Subpopulations

Over the decades since hematopoietic stem cells (HSCs) were first identified, the traditional view has been that the hematopoietic system is regenerated by a single pool of multipotent, quiescent HSCs that are sequentially recruited into cell cycle and which then progressively divide and differentiate until they are exhausted and ultimately replaced by the next cohort of stem cells. However, recent evidence has challenged this classical clonal succession model of HSC hierarchy by suggesting that the hematopoietic system is maintained by a pool of different HSC subtypes, with distinct self-renewal and differentiation potentials (the clonal diversity model, Figure 1). The side population (SP), characterized by Hoechst dye efflux, has been used as a method for isolating HSCs for over a decade and the SP has been shown to be highly enriched for HSC activity. While the entire SP is strikingly homogeneous with respect to expression of canonical stem cell markers such as Sca-1 and c-Kit, we recently observed heterogeneous expression for the SLAM family molecule CD150 within the SP, with CD150+ cells more prevalent in the lower SP and CD150− cell more prevalent in the upper SP. We decided to examine this observation further by investigating the properties of cells from different regions of the SP. Functional capacity was assessed by competitive bone marrow transplantation of upper SP cells, lower SP cells, and a combination of the two populations. Lower SP cells showed better engraftment than upper SP cells in recipient mice, a trend that continued when donor HSCs were isolated from primary recipients and re-transplanted into secondary hosts. Lower SP cells showed 3-fold better engraftment than upper SP cells in secondary transplants, suggesting better self-renewal capacity. However, analysis of the hematopoietic lineages formed by donor cells in recipient mice demonstrated that while both upper and lower SP cells were capable of forming all mature lineages, lower SP cells were biased towards myeloid differentiation while upper SP cells were biased towards lymphoid differentiation. The lineage biases observed from transplantation of one cell population alone were exacerbated when both upper and lower SP cells were co-transplanted into the same recipient mouse, suggesting that while both populations are capable of forming all hematopoietic lineages, in the presence of the other stem cell type (as would be the case in normal homeostasis) that the majority of the output from each HSC subtype is almost exclusively lymphoid or myeloid. The lineage contribution trends observed in the peripheral blood were also reproduced when bone marrow of transplanted mice was analyzed, including at the level of progenitors with lower SP cells showing greater ability to make myeloid progenitors (megakaryocyte-erythrocyte progenitors and granulocyte-macrophage progenitors) and upper SP cells producing proportionately more common lymphoid progenitors. Microarray analysis of upper and lower SP cells to determine the molecular signatures underlying these functional differences found many genes critical for long-term HSC self-renewal to be highly expressed in lower SP cells including Rb1, Meis1, Pbx1 and TGFbr2 while upper SP cells showed higher expression of cell cycle and activation genes. Cell cycle analysis showed upper SP cells to be approximately 2-fold more proliferative than lower SP cells (18.9% to 8.3% Ki-67+, 39.4% to 20.1% BrdU+ 3-days post-BrdU administration). The clonal diversity model which proposes the adult HSC compartment consists of a fixed number of different HSC subtypes each with pre-programmed behavior has important implications for using HSCs in experimental and clinical settings. While other studies have provided functional evidence for the clonal diversity model, this is the first study to prospectively isolate the functionally distinct HSC subtypes prior to transplantation. [Display omitted]

Copy Number Variant Analysis of Human Embryonic Stem Cells

Differences between individual DNA sequences provide the basis for human genetic variability. Forms of genetic variation include single-nucleotide polymorphisms, insertions/duplications, deletions, and inversions/translocations. The genome of human embryonic stem cells (hESCs) has been characterized mainly by karyotyping and comparative genomic hybridization (CGH), techniques whose relatively low resolution at 2-10 megabases (Mb) cannot accurately determine most copy number variability, which is estimated to involve 10%-20% of the genome. In this brief technical study, we examined HSF1 and HSF6 hESCs using array-comparative genomic hybridization (aCGH) to determine copy number variants (CNVs) as a higher-resolution method for characterizing hESCs. Our approach used five samples for each hESC line and showed four consistent CNVs for HSF1 and five consistent CNVs for HSF6. These consistent CNVs included amplifications and deletions that ranged in size from 20 kilobases to 1.48 megabases, involved seven different chromosomes, were both shared and unique between hESCs, and were maintained during neuronal stem/progenitor cell differentiation or drug selection. Thirty HSF1 and 40 HSF6 less consistently scored but still highly significant candidate CNVs were also identified. Overall, aCGH provides a promising approach for uniquely identifying hESCs and their derivatives and highlights a potential genomic source for distinct differentiation and functional potentials that lower-resolution karyotype and CGH techniques could miss. Disclosure of potential conflicts of interest is found at the end of this article.

Distinct osteoblastic differentiation potential of murine fetal liver and bone marrow stroma‐derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSC) are able to differentiate into osteoblasts under appropriate induction. Although MSC-derived osteoblasts are part of the hematopoietic niche, the nature of the stromal component in fetal liver remains elusive. Here, we determined the in vitro osteoblastic differentiation potential of murine clonal fetal liver-derived cells (AFT024, BFC012, 2012) in comparison with bone marrow-derived cell lines (BMC9, BMC10). Bone morphogenetic protein-2 (BMP2) increased alkaline phosphatase (ALP) activity, an early osteoblastic marker, in AFT024 and 2012 cells, whereas dexamethasone had little or no effect. BMP2, but not dexamethasone, increased ALP activity in BMC9 cells, and both inducers increased ALP activity in BMC10 cells. BMP2 increased ALP mRNA in AFT024, 2012 and BMC9 cells. By contrast, ALP was not detected in BMC10 and BFC012 cells. BMP2 and dexamethasone increased osteopontin and osteocalcin mRNA expression in 2012 cells. Furthermore, bone marrow-derived cells showed extensive matrix mineralization, whereas fetal liver-derived cell lines showed no or very limited matrix mineralization capacity. These results indicate that the osteoblast differentiation potential differs in bone marrow and fetal liver-derived cell lines, which may be due to a distinct developmental program or different microenvironment in the two hematopoietic sites.

Integrative genomic and functional analyses reveal neuronal subtype differentiation bias in human embryonic stem cell lines

The self-renewal and differentiation potential of human embryonic stem cells (hESCs) suggests that hESCs could be used for regenerative medicine, especially for restoring neuronal functions in brain diseases. However, the functional properties of neurons derived from hESC are largely unknown. Moreover, because hESCs were derived under diverse conditions, the possibility arises that neurons derived from different hESC lines exhibit distinct properties, but this possibility remains unexplored. To address these issues, we developed a protocol that allows stepwise generation from hESCs of cultures composed of approximately 70-80% human neurons that exhibit spontaneous synaptic network activity. Comparison of neurons derived from the well characterized HSF1 and HSF6 hESC lines revealed that HSF1- but not HSF6-derived neurons exhibit forebrain properties. Accordingly, HSF1-derived neurons initially form primarily GABAergic synaptic networks, whereas HSF6-derived neurons initially form glutamatergic networks. microRNA profiling revealed significant expression differences between the two hESC lines, suggesting that microRNAs may influence their distinct differentiation properties. These observations indicate that although both HSF1 and HSF6 hESCs differentiate into functional neurons, the two hESC lines exhibit distinct differentiation potentials, suggesting that they are preprogrammed. Information on hESC line-specific differentiation biases is crucial for neural stem cell therapy and establishment of novel disease models using hESCs.

Distinct Differentiation Potential of Blood Monocyte Subsets in the Lung

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1(high)CX(3)CR1(int) and Gr1(low)CX(3)CR1(high), respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1(low)CX(3)CR1(high) monocytes, but not Gr1(high)CX(3)CR1(int) cells, had the potential to differentiate into lung macrophages. However, Gr1(high)CX(3)CR1(int) monocytes could acquire this potential upon conversion into Gr1(low)CX(3)CR1(high) cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.

Identification of Cord Blood-Derived Mesenchymal Stem/stromal Cell Populations with Distinct Growth Kinetics, Differentiation Potentials, and Gene Expression Profiles

Phenotypic heterogeneity has been observed among mesenchymal stem/stromal cell (MSC) populations, but specific genes associated with this variability have not been defined. To study this question, we analyzed two distinct isogenic MSC populations isolated from umbilical cord blood (UCB1 and UCB2). The use of isogenic populations eliminated differences contributed by genetic background. We characterized these UCB MSCs for cell morphology, growth kinetics, immunophenotype, and potential for differentiation. UCB1 displayed faster growth kinetics, higher population doublings, and increased adipogenic lineage differentiation compared to UCB2. However, osteogenic differentiation was stronger for the UCB2 population. To identify MSC-specific genes and developmental genes associated with observed phenotypic differences, we performed expression analysis using Affymetrix microarrays and compared them to bone marrow (BM) MSCs. We compared UCB1, UCB2, and BM and identified distinct gene expression patterns. Selected clusters were analyzed demonstrating that genes of multiple developmental pathways, such as transforming growth factor-beta (TGF-beta) and wnt genes, and markers of early embryonic stages and mesodermal differentiation displayed significant differences among the MSC populations. In undifferentiated UCB1 cells, multiple genes were significantly up-regulated (p < 0.0001): peroxisome proliferation activated receptor gamma (PPARG), which correlated with adipogenic differentiation capacities, hepatocyte growth factor (HGF), and stromal-derived factor 1 (SDF1/CXCL12), which could both potentially contribute to the higher growth kinetics observed in UCB1 cells. Overall, the results confirmed the presence of two distinct isogenic UCB-derived cell populations, identified gene profiles useful to distinguish MSC types with different lineage differentiation potentials, and helped clarify the heterogeneity observed in these cells.

Intra–bone marrow transplantation facilitates pauci-clonal human hematopoietic repopulation of NOD/SCID/β2m −/− mice

Intra-bone marrow transplantation (IBMT) has been shown to improve the limit of detection of primitive human SCID-repopulating cells (SRC) in NOD/SCID mice when compared to intravenous transplantation. We sought to further refine detection of SRC by comparing NOD/SCID mice to the more sensitive NOD/SCID/beta2m(-/-)strain as IBMT recipients of limiting numbers of purified primitive human hematopoietic cells. Purified human Lin(-)CD34(+)CD38- cells at limiting doses were delivered by IBMT into NOD/SCID and NOD/SCID/beta2m(-/-) strains of recipient mice. Six weeks posttransplantation, injected and noninjected bones were analyzed separately for multilineage human hematopoietic chimerism. NOD/SCID/beta2m(-/-) mice are superior recipients for IBMT and show a trend toward increased levels of human hematopoietic engraftment. In addition, in contrast to NOD/SCID recipients, NOD/SCID/beta2m(-/-) mice were reconstituted with as few as five highly purified cells, indicative of pauci-clonal repopulation. Analysis of injected and noninjected bones demonstrated that engrafting cells were capable of in vivo migration and expansion. Although SRC hematopoietic reconstitution of NOD/SCID mice is commonly lymphoid-dominant, multilineage analysis of separate bone sites following IBMT of purified cells revealed that a subset of mice was repopulated with a myeloid-dominant graft in at least one bone site, revealing that SRC are developmentally heterogeneous among Lin(-)CD34(+)CD38- cells and capable of distinct differentiation potential. IBMT into NOD/SCID/beta2m(-/-) mice provides a highly sensitive experimental transplantation assay for the detection of human hematopoietic repopulating cells and demonstrates that Lin(-)CD34(+)CD38- cells are more highly enriched for human SRC than originally predicted.

N-methyl- N-nitrosourea-induced and spontaneous AKR/J thymic lymphomas express distinct differentiation antigen phenotypes

Spontaneous and N-methyl- N-nitrosourea (MNU)-induced AKR/J thymic lymphomas were characterized for expression of several lymphocyte differentiation antigens. The majority (53%) of spontaneous lymphomas expressed both Lyt-2 and L3T4 antigens, similar to the predominant normal thymocyte subset. In contrast, 63% of the thymic lymphomas induced by the chemical carcinogen MNU, expressed an Lyt-2+ L3T4- antigenic profile. Although this profile suggested that MNU-induced lymphomas are phenotypically similar to a mature thymocyte subset, the presence of ThB antigen on Lyt-2+ L3T4- lymphomas did not support this notion. Diagonal gel electrophoresis of 125I-labeled membrane extracts and immunoprecipitates revealed that 17 of 29 Lyt-2+ L3T4-MNU-induced lymphomas expressed cell surface T-cell receptor heterodimer components. Northern blot analyses confirmed that the T-cell receptor material was composed of α and β polypeptide chains. The results from this study indicate a distinct origin or differentiation potential of the target cells involved in viral and chemical induced lymphomagenesis of AKR/J mice.