Mesenchymal Stem Cells from Wharton's Jelly and Amniotic Fluid

The discovery of mesenchymal stem cells (MSCs) in perinatal sources, such as the amniotic fluid (AF) and the umbilical connective tissue, the so-called Wharton's jelly (WJ), has transformed them into promising stem cell grafts for the application in regenerative medicine. The advantages of AF-MSCs and WJ-MSCs over adult MSCs, such as bone marrow-derived mesenchymal stem cells (BMMSCs), include their minimally invasive isolation procedure, their more primitive cell character without being tumourigenic, their low immunogenicity and their potential autologous application in congenital disorders and when cryopreserved in adulthood. This chapter gives an overview of the biology of AF-MSCs and WJMSCs, and their regenerative potential based on the results of recent preclinical and clinical studies. In the end, open questions concerning the use of WJ-MSCs and AF-MSCs in regenerative medicine will be emphasized.

Successful transnasal delivery of stem cells in a rat model of perinatal hypoxic-ischemic brain injury

OBJECTIVE: New routes for cell transplantation into the brain need to be explored as intracerebral or intrathecal applications have a high risk to cause damage to the central nervous system. It has been hypothesized that transnasally administrated cells bypass the blood-brain barrier and migrate along the olfactory neural route into the brain and cerebrospinal fluid. Our goal is to confirm this hypothesis by transnasally administrating Wharton’s Jelly mesenchymal stem cells (WJ-MSC) and neural progenitor cells (NPC) to perinatal rats in a model of hypoxic-ischemic brain injury. STUDY DESIGN: Four-day-old Wistar rat pups, previously brain-damaged by combined hypoxic-ischemic and inflammatory insult, either received WJ-MSC or green fluorescent protein-expressing NPC: The heads of the rat pups were immobilized and 3 ml drops containing the cells (50’000 cells/ml) were placed on one nostril allowing it to be snorted. This procedure was repeated twice, alternating right to left nostril with an interval of one minute between administrations. The rat pups received a total of 600’000 cells. Animals were sacrificed 24h, 48h or 7 days after the application of the cells. Fixed brains were collected, embedded in paraffin and sectioned. RESULTS: Transplanted cells were found in the layers of the olfactory bulb (OB), the cerebral cortex, thalamus and the hippocampus. The amount of cells was highest in the OB. Animals treated with transnasally delivered stem cells showed significantly decreased gliosis compared to untreated animals. CONCLUSION: Our data show that transnasal delivery of WJ-MSC and NPC to the newborn brain after perinatal brain damage is successful. The cells not only migrate the brain, but also decrease scar formation and improve neurogenesis. Therefore, the non-invasive intranasal delivery of stem cells to the brain may be the preferred method for stem cell treatment of perinatal brain damage and should be preferred in future clinical trials.

Assessment of bovine adipose-derived stem cells osteogenic and adipogenic differentiation in Normoxic and hypoxic conditions

Background: The differentiation of ADSC is regulated by many factors, including oxygen tensions. Evidences have suggested that low oxygen tension or hypoxia is involved in the osteogenic, adipogenic differentiations of MSCs. Expansion and induction of ADSCs under hypoxia generally result in enhanced osteogenic, differentiation. Therefore, we analyzed bovine ADSC differentiations in Normoxia and hypoxia conditions Methodology: Recently (<8h) cow tail from a slaughterhouse, take out some fat from the tail and fat cells was isolated by using for isolation of ADSC protocol, the expansion cells were put into osteogenic and adipogenic medium for 3 weeks in hypoxia and normoxia conditions separately and characterized by Von kossa, Alizarin red and Oil red O staining and further by using real-time PCR by using primers of osteocalcin, Collagen type1, cbfa1/runx2, ALP, ostepontin, osteonectin, BMP2, BMP24, BMP27, noggin, gremlin, Nestin and HIF1A,VEGFA,PPARG,Leptin. Results: Our experiment results show hypoxia promotes osteogenesis but suppresses adipogenesis.

Dual Role of Mesenchymal Stem Cells Allows for Microvascularized Bone Tissue-Like Environments in PEG Hydrogels.

In vitro engineered tissues which recapitulate functional and morphological properties of bone marrow and bone tissue will be desirable to study bone regeneration under fully controlled conditions. Among the key players in the initial phase of bone regeneration are mesenchymal stem cells (MSCs) and endothelial cells (ECs) that are in close contact in many tissues. Additionally, the generation of tissue constructs for in vivo transplantations has included the use of ECs since insufficient vascularization is one of the bottlenecks in (bone) tissue engineering. Here, 3D cocultures of human bone marrow derived MSCs (hBM-MSCs) and human umbilical vein endothelial cells (HUVECs) in synthetic biomimetic poly(ethylene glycol) (PEG)-based matrices are directed toward vascularized bone mimicking tissue constructs. In this environment, bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2) promotes the formation of vascular networks. However, while osteogenic differentiation is achieved with BMP-2, the treatment with FGF-2 suppressed osteogenic differentiation. Thus, this study shows that cocultures of hBM-MSCs and HUVECs in biological inert PEG matrices can be directed toward bone and bone marrow-like 3D tissue constructs.

MacroH2A1 Regulation During the Cell Cycle of Mouse Embryonic Stem Cells

MacroH2A is a core histone variant that plays an important role in the X-inactivation process during differentiation of embryonic stem cells. It has been shown that macroH2A changes in localization during the cell cycle of somatic cells. This study aims to determine how macroH2A changes during the cell cycle of embryonic stem cells. Male and female mouse embryonic stem cells were transfected with a GFP::macroH2A construct and the relationship between macroH2A and the cell cycle was determined using FACS. This study shows that macroH2A is altered during the cell cycle of embryonic stem cells as it is in somatic cells and that in randomly cycling cells, there is a correlation between macroH2A expression and the phases of the cell cycle. High GFP expressing cells are mostly in the G2/M phase and low GFP expressing cells are mostly in the G1 phase. This correlation indicated that macroH2A is replicated with cellular DNA during the S phase resulting in higher expression in the G2/M phase. Future research, such as RT-PCR and differentiation experiments, is needed to further study this relationship and determine whether this change is at the protein or RNA level and how it changes during differentiation.

Determination of the Myogenic Potential of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells

Human embryonic stem cells (hESCs) have the potential to differentiate to all adult somatic cells. This property makes hESCs a very promising area of research for the treatment of disorders in which specific cell populations need to be restored. Despite this potential, research that focuses on producing mesodermally derived cell populations from hESCs is decidedly limited, notwithstanding the prevalence of disorders involving mesodermal tissues for which treatment options are limited. Skeletal muscle myoblasts are derivatives of mesodermal cells and are characterized by the expression of the MyoD gene. These cells are difficult to obtain from hESCs in a reproducible and efficient manner. Recent developments in the field have showed some success in obtaining myogenic cells from hESCs through a mesenchymal stem cell (MSC)-like intermediate population. MSCs, which are an adult stem cell population typically derived from the bone marrow, are capable of generating multiple cell types including skeletal muscle. The aim of this study was to develop an efficient method that derives myoblasts from an MSC-like intermediate. To accomplish this goal, we first set out to isolate and expand the MSC-like intermediate from hESCs differentiated in vitro. Difficulties in reproducing published cell-differentiation methodologies, which represent a significant and familiar challenge in hESC research, are highlighted in this report.

Potential REST Targets in Neural Stem Cells and Glioblastoma-Derived Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. The current theory is that these tumors are caused by self-renewing glioblastoma-derived stem cells (GSCs). At the current time, the mechanisms that regulate self-renewal and other oncogenic properties of GSCs remain unknown. Recently, we found transcriptional repressor REST maintains self-renewal in neural stem cells (NSCs) and in GSCs. REST also regulates other oncogenic properties, such as apoptosis, invasion and proliferation. However, the mechanisms by which REST regulates these oncogenic properties are unknown. In an attempt to determine these mechanisms, we performed loss and gain-of-function experiments and genome-wide mRNA expression analysis in GSCs, and we were able to identify REST-regulated genes in GSCs. This was accomplished, after screening concordantly regulated genes in NSCs and GSCs, utilizing two RE1 databases, and setting two-fold expression as filters on the resulting genes. These results received further validation by qRT-PCR. Ingenuity Pathway Analysis (IPA) analysis further revealed the top REST target genes in GSCs were downstream targets of REST and/or involved in other cancers in other cell lines. IPA also revealed that many of the differentially-regulated genes identified in this study are involved in oncogenic properties seen in GBM, and which we believe are related to REST expression.

PROSTATE CANCER STEM CELLS: DRUG TOLERANCE, EXPERIMENTAL RECONSTITUTION AND CASTRATION RESISTANCE

Prostate cancer (PCa) is one of the leading malignancies affecting men in the Western world. Although tremendous effort has been made towards understanding PCa development and developing clinical treatments in the past decades, the exact mechanisms of PCa are still not clearly understood. Emerging evidence has postulated that a population of stem cell-like cells inside a tumor, termed ‘cancer stem cells (CSCs)’, may be the cells responsible for tumor initiation, progression, recurrence, metastasis and therapy resistance. Like CSC studies in other cancer types, it has been reported that PCa also contains CSCs. However, there remain several unresolved questions that need to be clarified. First, the relationship between prostate CSCs (PCSCs) and therapy resistance (chemo- and radio-) is not known. Herein, we have found that not all CSCs are drug-tolerant, and not all drug-tolerant cells are CSCs. Second, whether primary human PCa (HPCa) actually contain PCSCs remains unclear, due to the well-known fact that we have yet to establish a reliable assay system that can reproducibly and faithfully reconstitute tumor regeneration from single HPCa cells. Herein, after utilizing more than 114 HPCa samples we have provided evidence that immortalized bone marrow-derived stromal cells (Hs5) can help dissociated HPCa cells generate undifferentiated tumors in immunodeficient NOD/SCID-IL2Rγ-/- mice, and the undifferentiated PCa cells seem to have a survival advantage to generate tumors. Third, the evolution of PCa from androgen dependent to the lethally castration resistant (CRPC) stage remains enigmatic, and the cells responsible for CRPC development have not been identified. Herein, we have found a putative cell population, ALDH+CD44+α2β1+ PCa cells that may represent a cell-of-origin for CRPC. Taken together, our work has improved our understanding of PCSC properties, possibly highlighting a potential therapeutic target for CRPC.

INVESTIGATING THE ROLES OF THE p63 ISOFORMS IN THE MICRORNA BIOGENESIS PATHWAY

MicroRNAs play roles in various biological processes like development, tumorigenesis, metastasis and pluripotency. My thesis work has demonstrated roles for p63, a p53 family member, in the upstream regulation of microRNA biogenesis. The p63 gene has a complex gene structure and has multiple isoforms. The TAp63 isoforms contain an acidic transcription activation domain. The ΔNp63 isoforms, lack the TA domain, but have a proline rich region critical for gene transactivation. To understand the functions of these isoforms, the Flores lab generated TAp63 and ΔNp63 conditional knock out mice. Using these mice and tissues and cells from these mice we have found that TAp63 transcriptionally regulates Dicer while ΔNp63 transcriptionally regulates DGCR8. TAp63 -/- mice are highly tumor prone. These mice develop metastatic mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas to distant sites including the liver, lungs, and brain. I found that TAp63 suppresses metastasis by transcriptionally activating Dicer. TAp63 and Dicer levels were very low or lost in high grade human tumors like mammary adenocarcinomas, squamous cell carcinomas, and lung adenocarcinomas. Expression of Dicer in these tumor cell lines reduced their invasiveness. Using ΔNp63 -/- mice, I found that ΔNp63 transcriptionally activates DGCR8, resulting in a miRNA profile that is critical to reprogram cells to pluripotency. Analysis of epidermal cells derived from ΔNp63 -/- mice revealed that these cells expressed markers of pluripotency, including Sox2, Oct 4 and Nanog; however, genome-wide analysis revealed a novel profile of genes that are common between ΔNp63 -/- epidermal cells and embryonic stem cells. I also found that mouse cells depleted of ΔNp63 form chimeric mice and teratomas in SCID mice, demonstrating that ΔNp63 deficient cells are pluripotent. Further, I found that restoration of DGCR8 in ΔNp63 -/- epidermal cells reduces their pluripotency and induces terminal differentiation. I also demonstrated that iMS (induced multipotent stem) cells could be generated using human keratinocytes by knockdown of ∆Np63 or DGCR8. Taken together, my work has placed p63 and its isoforms at a critical node in controlling miRNA biogenesis.

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression

Use of Echogenic Immunoliposomes for Delivery of both Drug and Stem Cells for Inhibition of Atheroma Progression By Ali K. Naji B.S. Advisor: Dr. Melvin E. Klegerman PhD Background and significance: Echogenic liposomes can be used as drug and cell delivery vehicles that reduce atheroma progression. Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis. VEGF functionally induces migration and proliferation of endothelial cells and increases intracellular vascular permeability. VEGF activates angiogenic transduction factors through VEGF tyrosine kinase domains in high-affinity receptors of endothelial cells. Bevacizumab is a humanized monoclonal antibody specific for VEGF-A which was developed as an anti-tumor agent. Often, anti-VEGF agents result in regression of existing microvessels, inhibiting tumor growth and possibly causing tumor shrinkage with time. During atheroma progression neovasculation in the arterial adventitia is mediated by VEGF. Therefore, bevacizumab may be effective in inhibiting atheroma progression. Stem cells show an ability to inhibit atheroma progression. We have previously demonstrated that monocyte derived CD-34+ stem cells that can be delivered to atheroma by bifunctional-ELIP ( BF-ELIP) targeted to Intercellular Adhesion Molecule-1 (ICAM-1) and CD-34. Adhesion molecules such as ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1) are expressed by endothelial cells under inflammatory conditions. Ultrasound enhanced liposomal targeting provides a method for stem cell delivery into atheroma and encapsulated drug release. This project is designed to examine the ability of echogenic liposomes to deliver bevacizumab and stem cells to inhibit atheroma progression and neovasculation with and without ultrasound in vitro and optimize the ultrasound parameters for delivery of bevacizumab and stem cells to atheroma. V Hypotheses: Previous studies showed that endothelial cell VEGF expression may relate to atherosclerosis progression and atheroma formation in the cardiovascular system. Bevacizumab-loaded ELIP will inhibit endothelial cell VEGF expression in vitro. Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP. I will also test the hypothesis that the transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma. Monocyte preparations will serve as a source of CD34+ stem cells. Specific Aims: Induce VEGF expression using PKA and PKC activation factors to endothelial cell cultures and use western blot and ELISA techniques to detect the expressed VEGF.  Characterize the relationship between endothelial cell proliferation and VEGF expression to develop a specific EC culture based system to demonstrate BEV-ELIP activity as an anti-VEGF agent. Design a cell-based assay for in vitro assessment of ultrasound-enhanced bevacizumab release from echogenic liposomes.  Demonstrate ultrasound delivery enhancement of stem cells by applying different types of liposomes on transwell EC culture using fluorescently labeled monocytes and detect the effect on migration and attachment rate of these echogenic liposomes with and without ultrasound in vitro.

Connexin-43 in the osteogenic BM niche regulates its cellular composition and the bidirectional traffic of hematopoietic stem cells and progenitors

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals

On a mathematical model of tumor growth based on cancer stem cells

We consider a simple mathematical model of tumor growth based on cancer stem cells. The model consists of four hyperbolic equations of first order to describe the evolution of different subpopulations of cells: cancer stem cells, progenitor cells, differentiated cells and dead cells. A fifth equation is introduced to model the evolution of the moving boundary. The system includes non-local terms of integral type in the coefficients. Under some restrictions in the parameters we show that there exists a unique homogeneous steady state which is stable.

Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in β1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1–27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, β-galactosidase (β-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3–33.3% of the colonies thus formed were β-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were β-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Hemato-lymphoid in vivo reconstitution potential of subpopulations derived from in vitro differentiated embryonic stem cells

During differentiation in vitro, embryonic stem (ES) cells generate progenitors for most hemato-lymphoid lineages. We studied the developmental potential of two ES cell subpopulations that share the fetal stem cell antigen AA4.1 but differ in expression of the lymphoid marker B220 (CD45R). Upon transfer into lymphoid deficient mice, the B220+ population generated a single transient wave of IgM+ IgD+ B cells but failed to generate T cells. In contrast, transfer of the B220− fraction achieved long-term repopulation of both T and B lymphoid compartments and restored humoral and cell-mediated immune reactions in the recipients. To assess the hemato-lymphopoietic potential of ES cell subsets in comparison to their physiological counterparts, cotransplantation experiments with phenotypically homologous subsets of fetal liver cells were performed, revealing a more potent developmental capacity of the latter. The results suggest that multipotential and lineage-committed lymphoid precursors are generated during in vitro differentiation of ES cells and that both subsets can undergo complete final maturation in vivo.