Turning Trash Into Treasure: A Simple Protocol for Human Mesenchymal Stromal Cell Isolation From Used Bone Marrow Collection Kits.

The therapeutic potential of mesenchymal stromal cells (MSCs) has been extensively investigated in both preclinical and clinical settings. Recent years have witnessed the emergence of numerous isolation protocols and culture techniques, ranging from the selection of subpopulations to preserve stemness to preconditioning strategies aimed at enhancing therapeutic efficacy, tailored to the specific tissue source. In this protocol, we present a straightforward and cost-effective method for isolating human MSCs (hMSCs) from discarded bone marrow collection kits (comprising bag and filter systems) originally intended for removing impurities and unwanted cellular debris from the collected bone marrow aspirate, ensuring the purity of the stem cell population during stem cell transplantation. Utilizing basic laboratory equipment, we demonstrate the isolation of hMSCs, highlighting the expression of specific surface antigens, and multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages in vitro. This sustainable and resource-efficient approach not only contributes to reducing medical waste but also holds promise for advancing regenerative medicine applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of human mesenchymal stromal cells from bone marrow collection kits Basic Protocol 2: Culture of human mesenchymal stromal cells Basic Protocol 3: Characterization of human mesenchymal stromal cells with flow cytometry analysis Basic Protocol 4: Characterization of human mesenchymal stromal cells with multilineage differentiation under in vitro conditions.

Investigation of androgen receptor gene CAG repeat length polymorphism in pubertal gynecomastia.

OBJECTIVES: Androgen receptor gene CAG repeat, AR (CAG)n, polymorphism is thought to have an effect on male reproductive functions and a relationship between long AR (CAG)n and decreased androgenic activity has been shown. Therefore, we hypothesized that in adolescents with long AR CAG repeat the prevalence of pubertal gynecomastia (PG) will be higher and we aimed to investigate the association between AR (CAG)n polymorphism and PG in Turkish adolescents. METHODS: Adolescents with PG between 11 and 19 years of age were enrolled as the study group and healthy individuals without a history of PG, who were at least 14 years of age and Tanner 4 or 5 were enrolled as the control group. The AR (CAG)n length was detected by direct DNA sequencing analysis and reproductive hormones were measured by standardized analyses. RESULTS: The mean AR (CAG)n was 22.3 ± 2.6 (mean ± SD) in the PG group (n=101) and 21.9 ± 3.1 (mean ± SD) in the control group (n=88) (p=0.276). The adolescents with short AR (CAG)n had lower body mass index standard deviation scores (BMI SDS) compared to the adolescents with intermediate and long repeat numbers (p=0.029). CONCLUSIONS: The results of this study showed a lack of direct association between AR (CAG)n and PG. However, the significant relationship between the AR (CAG)n quartiles and BMI SDS suggests that long AR (CAG)n might cause PG indirectly. Further studies are needed to better clarify this relationship.

Homozygous missense VPS16 variant is associated with a novel disease, resembling mucopolysaccharidosis-plus syndrome in two siblings.

Disorders of intracellular trafficking are a group of inherited disorders, which often display multisystem phenotypes. Vacuolar protein sorting (VPS) subunit C, composed of VPS11, VPS18, VPS16, and VPS33A proteins, is involved in tethering of endosomes, lysosomes, and autophagosomes. Our group and others have previously described patients with a specific homozygous missense VPS33A variant, exhibiting a storage disease phenotype resembling mucopolysaccharidosis (MPS), termed "MPS-plus syndrome." Here, we report two siblings from a consanguineous Turkish-Arabic family, who have overlapping features of MPS and intracellular trafficking disorders, including short stature, coarse facies, developmental delay, peripheral neuropathy, splenomegaly, spondylar dysplasia, congenital neutropenia, and high-normal glycosaminoglycan excretion. Whole exome sequencing and familial segregation analyses led to the homozygous NM_022575.3:c.540G>T; p.Trp180Cys variant in VPS16 in both siblings. Multiple bioinformatic methods supported the pathogenicity of this variant. Different monoallelic null VPS16 variants and a homozygous missense VPS16 variant had been previously associated with dystonia. A biallelic intronic, probably splice-altering variant in VPS16, causing an MPS-plus syndrome-like disease has been very recently reported in two individuals. The siblings presented herein display no dystonia, but have features of a multisystem storage disorder, representing a novel MPS-plus syndrome-like disease, associated for the first time with VPS16 missense variants.

Characterization of human bone marrow niches with metabolome and transcriptome profiling.

Bone marrow (BM) niches are special microenvironments that work in harmony with each other for the regulation and maintenance of hematopoiesis. Niche investigations have thus far been limited to various model organisms and animal studies; therefore, little is known about different niches in healthy humans. In this study, a special harvesting method for the collection of BM from two different anatomical regions in the iliac crest of humans was used to investigate the presence of different niches in BM. Additionally, metabolomic and transcriptomic profiles were compiled using comparative 'omics' technologies, and the main cellular pathways and corresponding transcripts and metabolites were identified. As a result, we found that the energy metabolism between the regions was different. This study provides basic broad data for regenerative medicine in terms of the design of the appropriate microenvironment for in vitro hematopoietic niche modeling, and identifies the normal reference values that can be compared in hematological disease.

A New Chapter for Mesenchymal Stem Cells: Decellularized Extracellular Matrices.

From orthopedic to neurological disorders, stem cells are used as platforms to understand disease mechanisms and considered as novel and promising treatment options, especially when the valid therapeutic approaches are unavailable or ineffective. There are different stem cell types in the literature, however the spindle-shaped, colony forming and multilineage-differentiating cells, also known as mesenchymal stem cells (MSC) are very popular, as MSC can be isolated from different tissues with minimal ethical concerns and without tumor formations, which make them easily accessible and widely used in vitro and in vivo studies. In the literature, MSC have been shown to have therapeutic effects and orchestrate the healing process via their mobilization, migration, differentiation capacities, immunomodulation properties and/or secretion of bioactive factors. Nowadays, MSC derived extracellular matrices (ECM), which are part of the secreted/produced bioactive molecules from MSC; draw attention of researchers due to their key roles in cell biology. Several groups have isolated ECM from in vitro cultured MSC using different methods of decellularization techniques for tissue-engineering approaches. According to current knowledge, decellularized ECM (dECM) influence growth, adhesion, differentiation, migration, apoptosis, proliferation, and phenotype of cells, covering almost all cellular events. In this comprehensive review we focused on MSC and the isolation methods and effects of MSC derived dECM (MSC-dECM).

Polymorphisms in FAS and CASP8 genes may contribute to the development of ALPS phenotype: a study in 25 patients with probable ALPS.

Defects in genes that have role in apoptotic pathways result in development of Autoimmune Lymphoproliferative Syndrome (ALPS) and ALPS related disorders. Germline and somatic FAS mutations, FASL and CASP10 mutations constitute other genetic defects in ALPS. Patients who fulfill ALPS diagnostic criteria and do not have any identified known disease causing mutations are classified as ALPS-unknown or ALPS phenotype and comprise about one third of all patients. CASP8, NRAS and KRAS gene mutations were reported for ALPS related diseases. We performed DNA sequence analysis in 25 unrelated patients with probable ALPS for FAS, FASL and CASP8 gene defects. Pathogenic mutations could not be found in the FAS, FASL and CASP8 genes. However, we found that the frequencies of SNPs rs2234978 and rs1045487 of FAS and CASP8 genes were significantly higher in the patients. Our results suggest that CASP8 and FAS gene polymorphisms in particular, may contribute to the susceptibility to development of ALPS phenotype.

MCP1 2518 A/G polymorphism affects progression of childhood focal segmental glomerulosclerosis.

Monocyte chemoattractant protein-1 (MCP-1) is a highly specific chemokine for monocytes and plays roles in pathogenesis of various renal diseases. The aim of this study is to investigate the effect of MCP1 2518 A/G polymorphism on the incidence and clinical course of focal segmental glomerulosclerosis (FSGS) in children. MCP1 2518 A/G genotype was identified by PCR-RFLP in 60 biopsy-proven FSGS patients, 76 steroid sensitive nephrotic syndrome (SSNS) patients, and 96 healthy children. MCP-1 levels in urine and serum were measured by ELISA in all patients and the correlations of genotype with MCP-1 levels and clinical outcome were evaluated. The genotype frequencies for MCP1 were similar in all groups. The percentage of patients who develop chronic renal failure was higher in patients with AA allele compared to GA or GG alleles (46% vs. 35% respectively, p < 0.01, Odds ratio: 1.59). Serum MCP-1 levels were similar in all groups, whereas urinary MCP-1 levels of the patients with FSGS (1680 pg/mg creatinine) were significantly higher than that of patients with SSNS (365 pg/mg creatinine, p < 0.05) and healthy controls (348 pg/mg creatinine; p < 0.05). Urinary MCP-1 levels were correlated with the degree of proteinuria in FSGS group (r = 0.529, p = 0.016). Our results suggest that the AA genotype might be a risk factor for the progression of renal disease in FSGS and MCP1 genotyping may help the physicians to predict prognosis in these patients.

Genome-wide homozygosity mapping in families with leber congenital amaurosis identifies mutations in AIPL1 and RDH12 genes.

Leber congenital amaurosis (LCA) causes severe visual impairment and blindness very early in life. Mutant alleles of several genes acting in different pathways, of which all have critical roles for normal retinal function, were involved in LCA development. The purpose of this study was to use genome-wide genotyping to identify LCA-causing loci in two Turkish families. Genome-wide genotyping and haplotype analysis were performed for prioritization of candidate genes for mutation screening in families with LCA. Identified informative critical choromosomal regions obtained by homozygosity mapping from the families were searched for overlapping of any LCA causative genes. Corresponding clinical phenotypes of the patients with identified mutations were evaluated. In this study, two families were shown to be linked to two different LCA loci covering retinol dehydrogenase 12 (RDH12) and aryl-hydrocarbon-interacting protein-like1 (AIPL1) genes. Mutation screening revealed a novel p.Gln141* mutation in the AIPL1 gene and a previously described p.Thr49Met mutation in the RDH12 gene in a homozygous state. Our patients with the RDH12 mutation had the distinct macular coloboma sign, and the patient with the AIPL1 mutation developed microphthalmia and severe widespread retinal pigment epithelial atrophy, in contrast to previously reported cases. It is currently evident that mutation screening needs to be done in at least 18 genes known to be associated with LCA. Thus, homozygosity mapping is an alternative technique to improve the molecular diagnosis in LCA, which is a group of genetically and clinically heterogeneous diseases causing retinal degeneration. The patients without mutation in known genes may further be analyzed by using next-generation sequencing.

High prevalence of cerebral venous sinus thrombosis (CVST) as presentation of cystathionine beta-synthase deficiency in childhood: molecular and clinical findings of Turkish probands.

Classical homocystinuria is the most commonly inherited disorder of sulfur metabolism, caused by the genetic alterations in human cystathionine beta-synthase (CBS) gene. In this study, we present comprehensive clinical findings and the genetic basis of homocystinuria in a cohort of Turkish patients. Excluding some CBS mutations, detailed genotype-phenotype correlation for different CBS mutations has not been established in literature. We aimed to make clinical subgroups according to main clinical symptoms and discussed these data together with mutational analysis results from our patients. Totally, 16 different mutations were identified; twelve of which had already been reported, and four are novel (p.N93Y, p.L251P, p.D281V and c.829-2A>T). The probands were classified into three major groups according to the clinical symptoms caused by these mutations. A psychomotor delay was the most common diagnostic symptom (n=12, 46.2% neurological presentation), followed by thromboembolic events (n=6, 23.1% vascular presentation) and lens ectopia, myopia or marfanoid features (n=5, 19.2% connective tissue presentation). Pyridoxine responsiveness was 7.7%; however, with partial responsive probands, the ratio was 53.9%. In addition, five thrombophilic nucleotide changes including MTHFR c.677 C>T and c.1298 A>C, Factor V c.1691 G>A, Factor II c.20210 G>A, and SERPINE1 4G/5G were investigated to assess their contributions to the clinical spectrum. We suggest that the effect of these polymorphisms on clinical phenotype of CBS is not very clear since the distribution of thrombophilic polymorphisms does not differ among specific groups. This study provides molecular findings of 26 Turkish probands with homocystinuria and discusses the clinical presentations and putative effects of the CBS mutations.

Dynamics of the rhomboid-like protein RHBDD2 expression in mouse retina and involvement of its human ortholog in retinitis pigmentosa.

The novel rhomboid-like protein RHBDD2 is distantly related to rhomboid proteins, a group of highly specialized membrane-bound proteases that catalyze regulated intramembrane proteolysis. In retina, RHBDD2 is expressed from embryonic stages to adulthood, and its levels show age-dependent changes. RHBDD2 is distinctly abundant in the perinuclear region of cells, and it localizes to their Golgi. A glycine zipper motif present in one of the transmembrane domains of RHBDD2 is important for its packing into the Golgi membranes. Its deletion causes dislodgment of RHBDD2 from the Golgi. A specific antibody against RHBDD2 recognizes two forms of the protein, one with low (39 kDa; RHBDD2(L)) and the other with high (117 kDa; RHBDD2H) molecular masses in mouse retinal extracts. RHBDD2(L) seems to be ubiquitously expressed in all retinal cells. In contrast, RHBDD2H seems to be present only in the outer segments of cone photoreceptors and may correspond to a homotrimer of RHBDD2(L). This protein consistently co-localizes with S- and M-types of cone opsins. We identified a homozygous mutation in the human RHBDD2 gene, R85H, that co-segregates with disease in affected members of a family with autosomal recessive retinitis pigmentosa. Our findings suggest that the RHBDD2 protein plays important roles in the development and normal function of the retina.

Cobalamin C defect: a patient of late-onset type with homozygous p.R132* mutation.

Methylmalonic aciduria and homocystinuria, cobalamin C (cblC) type, is the most frequent inborn error of vitamin B12metabolism. The clinical phenotype includes systemic symptoms and neurological decompensation. Affected patients can be divided into two broad groups, as early-onset and late-onset. We present a Turkish patient who had neurological impairment at the age of four years as presented with late-onset cblC defect. Homozygous c.394C