Marcin Jurga

Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro

This 3-week protocol produces embryonic-like stem cells from human umbilical cord blood (CBEs) for neural differentiation using a three-step system (cell isolation/expansion/differentiation). The CBE isolation produces a highly purified fraction (CD45−, CD33−, CD7−, CD235a−) of small pluripotent stem cells (2–3 μm in diameter) coexpressing embryonic stem cell markers including Oct4 and Sox2. Initial CBE expansion is performed in high density (5–10 millions per ml) in the presence of extracellular matrix proteins and epidermal growth factor. Subsequent neural differentiation of CBEs requires sequential introduction of morphogenes, retinoic acid, brain-derived neurotrophic factor and cyclic AMP. Described methods emphasize defined media and reagents at all stages of the experiment comparable to protocols described for culturing human embryonic stem cells and cells from other somatic stem cell sources. Neural progenitor and cells generated from CBEs may be used for in vitro drug testing and cell-based assays and potentially for clinical transplantation.

Promising New Sources for Pluripotent Stem Cells

Recent findings have placed stem cell research at the forefront of biomedical sciences. Basic research on embryonic stem cells (ESCs) has contributed to our knowledge about the developmental potential and plasticity of stem cells. Furthermore, it has raised hope to use these cells as potential source for regenerative medicine and tissue replacement after injury or disease. Unfortunately, ESCs can also form tumors and they are ethically controversial because they originate from human embryos. This review summarizes findings and therapeutic applications of ESCs and their alternatives: adult stem cells and iPS cells.

Neurogenic potential of human umbilical cord blood: Neural-like stem cells depend on previous long-term culture conditions

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB‐NPs). Due to further expansion of these cells we have established the first human umbilical cord blood‐derived neural‐like stem cell line (HUCB‐NSC) growing in serum‐free (SF) or low‐serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB‐NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB‐NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co‐cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co‐cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial‐specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB‐NSC on the neurogenic signals provided by surrounding environment in vitro.

Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner

BACKGROUND AIMS As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly-derived MSCs (WJ-MSCs). METHODS Primary cultures growing in 21% oxygen were either transferred into 5% O2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α-3α were evaluated. RESULTS Lowering O2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration. CONCLUSIONS A physiologically relevant microenvironment of 5% O2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α-mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.

New perspectives in stem cell research: beyond embryonic stem cells

Although stem cell research is a rather new field in modern medicine, media soon popularized it. The reason for this hype lies in the potential of stem cells to drastically increase quality of life through repairing aging and diseased organs. Nevertheless, the essence of stem cell research is to understand how tissues are maintained during adult life. In this article, we summarize the various types of stem cells and their differentiation potential in vivo and in vitro. We review current clinical applications of stem cells and highlight problems encountered when going from animal studies to clinical practice. Furthermore, we describe the current state of induced pluripotent stem cell technology and applications for disease modelling and cell replacement therapy.

Neuronal Differentiation of Human Umbilical Cord Blood Neural Stem-Like Cell Line

The expanding population of neural stem/progenitor cells can be selected from human cord blood nonhematopoietic (CD34-negative) mononuclear fraction. Due to repeated expansion and selection of these cells we have established the first clonogenic, nonimmortalized human umbilical cord blood neural stem-like cell (HUCB-NSC) line. This line can be maintained at different stages of neural progenitor development by the presence of trophic factors, mitogens and neuromorphogens in culture media. Neurogenic potential of HUCB-NSC was established for serum-free and low-serum cultured cells. Commitment of HUCB-NSC by serum was shown to be important for the optimal response to the signals provided by surrounding environment in vitro. Enhanced neuronal differentiation induced by dBcAMP treatment was accompanied by expression of several functional proteins including glutamatergic, GABAergic, dopamine, serotonin and acetylcholine receptors, which was shown by microarray, immunocytochemistry and electrophysiology. Electrophysiological studies, whole-cell patch-clamp recordings, revealed in differentiated HUCB-NSC two types of voltage-sensitive and several ligand-gated currents typical for neuronal cells. The above HUCB-NSC characteristic conceivably implicates that cord blood-derived progenitors could be effectively differentiated into functional neuron-like cells in vitro.

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Experimental therapies for repair of the central nervous system: stem cells and tissue engineering.

Several stem cell‐based therapeutic tools are currently being investigated for the regeneration of central nervous system (CNS) injuries. This review focuses on innovative approaches for CNS tissue repair via the use of implantable cellular devices. These devices are supported by biopharmaceuticals and conventional physiotherapy for the restoration of lost neuronal circuits and CNS function. This paper further reviews new and promising tools currently in pre‐clinical and clinical tests for the treatment of CNS diseases where substantial loss of cellular and extracellular components of neural tissue has occurred such as stroke, encephalopathy and traumatic neural injuries. We also discuss selected 3D bioscaffolds co‐cultured with clinically applicable human mesenchymal stem cells. Recent advances in neural tissue engineering and stem cell differentiation methods have shown promise for their clinical application in treating yet incurable CNS deficits. Copyright

Encapsulation of Mesenchymal Stem Cells by Bioscaffolds Protects Cell Survival and Attenuates Neuroinflammatory Reaction in Injured Brain Tissue after Transplantation

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin–laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Whartons jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the hosts immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

miRNAs Stem Cell Reprogramming for Neuronal Induction and Differentiation

Mimicking the natural brain environment during neurogenesis represents the main challenge for efficient in vitro neuronal differentiation of stem cells. The discovery of miRNAs opens new possibilities in terms of modulation of stem cells lineage commitment and differentiation. Many studies demonstrated that in vitro transient overexpression or inhibition of brain-specific miRNAs in stem cells significantly directed differentiation along neuronal cell lineages. Modulating miRNA expression offers new pathways for post-transcriptional gene regulation and stem cell commitment. Neurotrophins and neuropoietins signaling pathways are the main field of investigation for neuronal commitment, differentiation, and maturation. This review will highlight examples of crosstalk between stem-cell-specific and brain-specific signaling pathways and key miRNA candidates for neuronal commitment. Recent progress on understanding miRNAs genetic networks offers promising prospects for their increasing application in the development of new cellular therapies in humans.