搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Coronary heart disease (CHD) is a leading cause of death globally,while its current management is limited to reducing the myocardial infarction area without actually replacing dead cardiomyocytes. Direct cell reprogramming is a method of cellular cardiomyoplasty which aims for myocardial tissue regeneration,and CD34+ cells are one of the potential sources due to their shared embryonic origin with cardiomyocytes. However,the isolation and culture of non-adherent CD34+ cells is crucial to obtain adequate cells for high-efficiency genetic modification. This study aimed to investigate the optimal method for isolation and culture of CD34+ peripheral blood cells using certain culture media. A peripheral blood sample was obtained from a healthy subject and underwent pre-enrichment,isolation,and expansion. The culture was subsequently observed for their viability,adherence,and confluence. Day 0 observation of the culture showed a healthy CD34+ cell with a round cell shape,without any adherent cells present yet. Day 4 of observation showed that CD34+ cells within the blood plasma medium became adherent,indicated by their transformations into spindle or oval morphologies. Meanwhile,CD34+ cells in vitronectin and fibronectin media showed no adherent cells and many of them died. Day 7 observation revealed more adherent CD34+ cells in blood plasma medium,and which had 75% of confluence. In conclusion,the CD34+ cells that were isolated using a combination of density and magnetic methods may be viable and adequately adhere in culture using blood plasma medium,but not in cultures using fibronectin and vitronectin. View Publication

The considerable therapeutic potential of human multipotent mesenchymal stromal cells (MSC) has generated markedly increasing interest in a wide variety of biomedical disciplines. However,investigators report studies of MSC using different methods of isolation and expansion,and different approaches to characterizing the cells. Thus it is increasingly difficult to compare and contrast study outcomes,which hinders progress in the field. To begin to address this issue,the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposes minimal criteria to define human MSC. First,MSC must be plastic-adherent when maintained in standard culture conditions. Second,MSC must express CD105,CD73 and CD90,and lack expression of CD45,CD34,CD14 or CD11b,CD79alpha or CD19 and HLA-DR surface molecules. Third,MSC must differentiate to osteoblasts,adipocytes and chondroblasts in vitro. While these criteria will probably require modification as new knowledge unfolds,we believe this minimal set of standard criteria will foster a more uniform characterization of MSC and facilitate the exchange of data among investigators. View Publication

Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors,Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However,several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently,it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However,NPCs express Sox2 endogenously,possibly facilitating reprogramming in the absence of exogenous Sox2. In this study,we identified a small-molecule combination,BIX-01294 and BayK8644,that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts,which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors,such as Sox2,and improve reprogramming efficiency. View Publication

Innate lymphoid cells 2 (ILC2) play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma (PDAC). An important aspect of ILC2-mediated tumorigenesis is the expansion of the resident ILC2 and simultaneous recruitment of the peripheral ILC2. Here,we describe a protocol for isolation,enrichment,and DiD labeling of ILC2 for in vivo tracking of ILC2s in the mouse. Further,we describe steps for the adoptive transfer of ILC2 to a recipient mouse model of PDAC. For complete details on the use and execution of this protocol,please refer to Alam et al. (2022). View Publication

Angiotensin I-converting enzyme (ACE) inhibitors can affect hematopoiesis by several mechanisms including inhibition of angiotensin II formation and increasing plasma concentrations of AcSDKP (acetyl-N-Ser-Asp-Lys-Pro),an ACE substrate and a negative regulator of hematopoiesis. We tested whether ACE inhibition could decrease the hematopoietic toxicity of lethal or sublethal irradiation protocols. In all cases,short treatment with the ACE inhibitor perindopril protected against irradiation-induced death. ACE inhibition accelerated hematopoietic recovery and led to a significant increase in platelet and red cell counts. Pretreatment with perindopril increased bone marrow cellularity and the number of hematopoietic progenitors (granulocyte macrophage colony-forming unit [CFU-GM],erythroid burst-forming unit [BFU-E],and megakaryocyte colony-forming unit [CFU-MK]) from day 7 to 28 after irradiation. Perindopril also increased the number of hematopoietic stem cells with at least a short-term reconstitutive activity in animals that recovered from irradiation. To determine the mechanism of action involved,we evaluated the effects of increasing AcSDKP plasma concentrations and of an angiotensin II type 1 (AT1) receptor antagonist (telmisartan) on radioprotection. We found that the AT1-receptor antagonism mediated similar radioprotection as the ACE inhibitor. These results suggest that ACE inhibitors and AT1-receptor antagonists could be used to decrease the hematopoietic toxicity of irradiation. View Publication

Retrovirus-mediated transduction of Hoxb4 enhances hematopoietic stem cell (HSC) activity and enforced expression of Hoxb4 induces in vitro development of HSCs from differentiating mouse embryonic stem cells,but the underlying molecular mechanism remains unclear. We previously showed that the HSC activity was abrogated by accumulated Geminin,an inhibitor for the DNA replication licensing factor Cdt1 in mice deficient in Rae28 (also known as Phc1),which encodes a member of Polycomb-group complex 1. In this study we found that Hoxb4 transduction reduced accumulated Geminin in Rae28-deficient mice,despite increasing the mRNA,and restored the impaired HSC activity. Supertransduction of Geminin suppressed the HSC activity induced by Hoxb4 transduction,whereas knockdown of Geminin promoted the clonogenic and replating activities,indicating the importance of Geminin regulation in the molecular mechanism underlying Hoxb4 transduction-mediated enhancement of the HSC activity. This facilitated our investigation of how transduced Hoxb4 reduced Geminin. We showed in vitro and in vivo that Hoxb4 and the Roc1 (also known as Rbx1)-Ddb1-Cul4a ubiquitin ligase core component formed a complex designated as RDCOXB4,which acted as an E3 ubiquitin ligase for Geminin and down-regulated Geminin through the ubiquitin-proteasome system. Down-regulated Geminin and the resultant E2F activation may provide cells with proliferation potential by increasing a DNA prereplicative complex loaded onto chromatin. Here we suggest that transduced Hoxb4 down-regulates Geminin protein probably by constituting the E3 ubiquitin ligase for Geminin to provide hematopoietic stem and progenitor cells with proliferation potential. View Publication

Self-renewal is a complex biological process necessary for maintaining the pluripotency of embryonic stem cells (ESCs). Recent studies have used global proteomic techniques to identify proteins that associate with the master regulators Oct4,Nanog and Sox2 in ESCs or in ESCs during the early stages of differentiation. Through an unbiased proteomic screen,Banf1 was identified as a Sox2-associated protein. Banf1 has been shown to be essential for worm and fly development but,until now,its role in mammalian development and ESCs has not been explored. In this study,we examined the effect of knocking down Banf1 on ESCs. We demonstrate that the knockdown of Banf1 promotes the differentiation of mouse ESCs and decreases the survival of both mouse and human ESCs. For mouse ESCs,we demonstrate that knocking down Banf1 promotes their differentiation into cells that exhibit markers primarily associated with mesoderm and trophectoderm. Interestingly,knockdown of Banf1 disrupts the survival of human ESCs without significantly reducing the expression levels of the master regulators Sox2,Oct4 and Nanog or inducing the expression of markers of differentiation. Furthermore,we determined that the knockdown of Banf1 alters the cell cycle distribution of both human and mouse ESCs by causing an uncharacteristic increase in the proportion of cells in the G2-M phase of the cell cycle. View Publication

Human and mouse embryonic stem cells (ESCs) are derived from blastocyst-stage embryos but have very different biological properties,and molecular analyses suggest that the pluripotent state of human ESCs isolated so far corresponds to that of mouse-derived epiblast stem cells (EpiSCs). Here we rewire the identity of conventional human ESCs into a more immature state that extensively shares defining features with pluripotent mouse ESCs. This was achieved by ectopic induction of Oct4,Klf4,and Klf2 factors combined with LIF and inhibitors of glycogen synthase kinase 3beta (GSK3beta) and mitogen-activated protein kinase (ERK1/2) pathway. Forskolin,a protein kinase A pathway agonist which can induce Klf4 and Klf2 expression,transiently substitutes for the requirement for ectopic transgene expression. In contrast to conventional human ESCs,these epigenetically converted cells have growth properties,an X-chromosome activation state (XaXa),a gene expression profile,and a signaling pathway dependence that are highly similar to those of mouse ESCs. Finally,the same growth conditions allow the derivation of human induced pluripotent stem (iPS) cells with similar properties as mouse iPS cells. The generation of validated naïve" human ESCs will allow the molecular dissection of a previously undefined pluripotent state in humans and may open up new opportunities for patient-specific� View Publication

By retroviral overexpression of the Notch-1 intracellular domain (ICN) in human CD34+ hematopoietic stem cells (HSCs),we have shown previously that Notch-1 signaling promotes the T-cell fate and inhibits the monocyte and B-cell fate in several in vitro and in vivo differentiation assays. Here,we investigated whether the effects of constitutively active Notch-1 can be mimicked by overexpression of its downstream target gene HES1. Upon HES-1 retroviral transduction,human CD34+ stem cells had a different outcome in the differentiation assays as compared to ICN-transduced cells. Although HES-1 induced a partial block in B-cell development,it did not inhibit monocyte development and did not promote T/NK-cell-lineage differentiation. On the contrary,a higher percentage of HES-1-transduced stem cells remained CD34+. These experiments indicate that HES-1 alone is not able to substitute for Notch-1 signaling to induce T-cell differentiation of human CD34+ hematopoietic stem cells. View Publication

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6,and atypical protein kinase C (aPKCζ and aPKCλ),which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However,the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that,in contrast to accepted paradigms,polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice,having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ,respectively,have normal hematopoiesis,including normal HSC self-renewal,engraftment,differentiation,and interaction with the bone marrow microenvironment. Furthermore,inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization,self-renewal,engraftment,or lineage repopulation. In addition,aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together,the expression of aPKCζ,aPKCλ,or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis,contrary to the hypothesis of a unique,evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination. View Publication

Stem cells hold great promise for therapies aimed at regenerating damaged tissue,drug screening and studying in vitro models of human disease. However,many challenges remain before these applications can become a reality. One such challenge is developing chemically defined and scalable culture conditions for derivation and expansion of clinically viable human pluripotent stem cells,as well as controlling their differentiation with high specificity. Interaction of stem cells with their extracellular microenvironment plays an important role in determining their differentiation commitment and functions. Regenerative medicine approaches integrating cell-matrix and cell-cell interactions,and soluble factors could lead to development of robust microenvironments to control various cellular responses. Indeed,several of these recent developments have provided significant insight into the design of microenvironments that can elicit the targeted cellular response. In this article,we will focus on some of these developments with an emphasis on matrix-mediated expansion of human pluripotent stem cells while maintaining their pluripotency. We will also discuss the role of matrix-based cues and cell-cell interactions in the form of soluble signals in directing stem cell differentiation into musculoskeletal lineages. View Publication