Adam Nowakowski

Real-time MRI for precise and predictable intra-arterial stem cell delivery to the central nervous system

Stem cell therapy for neurological disorders reached a pivotal point when the efficacy of several cell types was demonstrated in small animal models. Translation of stem cell therapy is contingent upon overcoming the challenge of effective cell delivery to the human brain, which has a volume ∼1000 times larger than that of the mouse. Intra-arterial injection can achieve a broad, global, but also on-demand spatially targeted biodistribution; however, its utility has been limited by unpredictable cell destination and homing as dictated by the vascular territory, as well as by safety concerns. We show here that high-speed MRI can be used to visualize the intravascular distribution of a superparamagnetic iron oxide contrast agent and can thus be used to accurately predict the distribution of intra-arterial administered stem cells. Moreover, high-speed MRI enables the real-time visualization of cell homing, providing the opportunity for immediate intervention in the case of undesired biodistribution.

Genetic Engineering of Mesenchymal Stem Cells to Induce Their Migration and Survival

Mesenchymal stem cells (MSCs) are very attractive for regenerative medicine due to their relatively easy derivation and broad range of differentiation capabilities, either naturally or induced through cell engineering. However, efficient methods of delivery to diseased tissues and the long-term survival of grafted cells still need improvement. Here, we review genetic engineering approaches designed to enhance the migratory capacities of MSCs, as well as extend their survival after transplantation by the modulation of prosurvival approaches, including prevention of senescence and apoptosis. We highlight some of the latest examples that explore these pivotal points, which have great relevance in cell-based therapies.

Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine

Mesenchymal stem cells (MSCs), which can be obtained from various organs and easily propagated in vitro, are one of the most extensively used types of stem cells and have been shown to be efficacious in a broad set of diseases. The unique and highly desirable properties of MSCs include high migratory capacities toward injured areas, immunomodulatory features, and the natural ability to differentiate into connective tissue phenotypes. These phenotypes include bone and cartilage, and these properties predispose MSCs to be therapeutically useful. In addition, MSCs elicit their therapeutic effects by paracrine actions, in which the metabolism of target tissues is modulated. Genetic engineering methods can greatly amplify these properties and broaden the therapeutic capabilities of MSCs, including transdifferentiation toward diverse cell lineages. However, cell engineering can also affect safety and increase the cost of therapy based on MSCs; thus, the advantages and disadvantages of these procedures should be discussed. In this review, the latest applications of genetic engineering methods for MSCs with regenerative medicine purposes are presented.

Translation, but not transfection limits clinically relevant, exogenous mRNA based induction of alpha-4 integrin expression on human mesenchymal stem cells

Mesenchymal stem cells (MSCs) represent promising resource of cells for regenerative medicine in neurological disorders. However, efficient and minimally invasive methods of MSCs delivery to the brain still have to be developed. Intra-arterial route is very promising, but MSCs are missing machinery for diapedesis through blood-brain barrier. Thus, here we have tested a mRNA-based method to induce transient expression of ITGA4, an adhesion molecule actively involved in cell extravasation. We observed that transfection with an ITGA4-mRNA construct bearing a conventional cap analogue (7-methylguanosine) failed to produce ITGA4 protein, but exogenous ITGA4-mRNA was detected in transfected MSCs. This indicates that not transfection, but rather translation being the major roadblock. Stabilization of ITGA4-mRNA with SSB proteins resulted in ITGA4 protein synthesis in HEK293 cells only, whereas in MSCs, satisfactory results were obtained only after using an anti-reverse-cap-analogue (ARCA). The presence of ITGA4 protein in MSCs was transient and lasted for up to 24 h after transfection. Membranous location was confirmed by flow cytometry of viable non-permeabilized cells using anti-ITGA4 antibody. The mRNA-based expression of itga4 transgene is potentially sufficient for diapedesis after intra-arterial delivery. To conclude, mRNA-based engineering of stem cells is a rapid and integration-free method and attractive from the perspective of potential future clinical application.

Pre- and postmortem imaging of transplanted cells

Therapeutic interventions based on the transplantation of stem and progenitor cells have garnered increasing interest. This interest is fueled by successful preclinical studies for indications in many diseases, including the cardiovascular, central nervous, and musculoskeletal system. Further progress in this field is contingent upon access to techniques that facilitate an unambiguous identification and characterization of grafted cells. Such methods are invaluable for optimization of cell delivery, improvement of cell survival, and assessment of the functional integration of grafted cells. Following is a focused overview of the currently available cell detection and tracking methodologies that covers the entire spectrum from pre- to postmortem cell identification.

An axonal form of Charcot-Marie-Tooth disease with a novel missense mutation in the myelin protein zero gene

Dear Editor, Myelin protein zero (MPZ), a major component of peripheral myelin, is expressed by myelinating Schwann cells (Lemke et al., 1988). Simultaneously to studies on the role of MPZ protein in myelin maintenance, axonal development was shown to depend on proper function of the MPZ protein (Martini et al., 1995). Molecular genetic analysis of the MPZ gene confirmed the role of the MPZ protein not only in demyelinating but also in axonal forms of the Charcot-Marie-Tooth disease type 1 (CMT1) and CMT2. The MPZ gene was initially cloned from rat, and 3 years later, its human homolog was shown to map to the 1q21-q23 chromosome, the locus of CMT1B (Bird et al., 1982). The first mutation in the MPZ gene found in the CMT1B family supported a MPZ role in myelin sheath maintenance (Warner et al., 1996). The first Ser44 Phe mutation in the MPZ gene was reported in CMT2 disease in a large Sardinian family (Marrosu et al., 1999). We report a novel Gly56Lys mutation in the MPZ gene found in a third generation CMT2 family. According to the proband, her father, who died at the age of 70, was diagnosed with neuropathy and deafness of unknown origin. The proband’s sister revealed, on neurological examination at the age of 36 years, pes planus and thoraco-lumbar scoliosis only. The proband’s son at the age of 23 showed pes cavus and slim calf muscles. He did not give his informed consent for molecular genetic analysis. The proband, at the age of 41 years, started to complain of cramps in the lower limbs and difficulty in walking. On neurological examination (at age 44), ulnar and radial jerks were absent in the upper limbs and, in lower limbs, pronounced distal muscle atrophy and weakness as well as pes cavus were found; she could not walk on her heels. Mild thoracic scoliosis was present, and cranial nerves were normal and sensation preserved. Electrophysiological examination revealed that left median nerve motor conduction velocity (MCV) distally was 41.2m/s, proximally was 54.2m/s, and distal latency (DL) was 3.1ms. Sensory conduction velocity (SCV) was 57.4m/s, DL was 2.7ms, and amplitude of evoked potentials was 4.0 mV. In the peroneal nerve, MCV was 30.1m/s, DL was 5.0ms, amplitude of evoked potentials was 1.0mV, and F wave latency was 69.8. In sural nerve, SCV was 30.8m/s, DL was 6.0ms, and amplitude of evoked potentials was 2.9 mV. The SSCP analysis of the second exon of the MPZ gene revealed an altered migration pattern in the proband, her sister, and her son. In the sequencing of exon 2 of the MPZ gene, a heterozygous G-A transition (sense strand) and C-T substitution (antisense strand) was detected in the proband, her sister, and her son, resulting in a Glu to Lys amino acid substitution at codon 56 of the MPZ gene (Glu56Lys). The presented family suffers from mixed neuropathy with preponderance of axonal lesion, as judged by the electrophysiological study. Whether demyelination is primary or secondary to axonal lesion is not resolved. The obvious muscle atrophy of legs in comparison with the short duration of clinical symptoms in the proband incline us to include this family in the CMT2 population. Genetic analysis revealed Glu56Lys mutation in four members of the family. To the best of our knowledge, the reported Glu56Lys mutation is a new genetic defect in the MPZ gene segregating with the CMT2 phenotype. We cannot state whether, in our family, the onset of overt clinical symptoms is rather late or reflects the variable expression of the Gly56Lys mutation in the MPZ gene. Due to lack of experimental results, we can only hypothesize that the presently Address correspondence to: Andrzej Kochanski, Neuromuscular Unit, Medical Research Centre, Polish Academy of Sciences, Banacha 1a, 02-098 Warsaw, Poland. E-mail: andko@cmdik.pan.pl Journal of the Peripheral Nervous System 9:1–2 (2004)