Stem Cells and Development

Hematopoietic stem cells (HSCs) are multipotent stem cells capable of differentiating into all types of blood and immune progeny and endowed with self-renewal capacity to enable lifelong hematopoiesis. Based on these unique characteristics, HSCs are utilized as a cell source for cell and gene therapies. During its dawning period, HSC-based therapies faced significant challenges due to inefficient gene transfer and insertional leukemogenesis. However, technological advances, such as the use of HIV-derived lentiviral vectors and cellular promoters, have established HSC gene therapy as a powerful treatment modality for patients with congenital monogenic diseases, leading to approved therapies commercially available in Europe and the United States. HSC-based therapies are now being explored for broader indications, including cancer, autoimmune, and infectious diseases. Innovative concepts achievable with HSCs—such as delivering therapeutic proteins to hard-to-reach tissues, in vivo delivery of antibodies and immune cells, and molecular shielding—have been proposed, offering new therapeutic approaches. Moreover, technological innovations in related fields, including more precise gene expression control and reduced-toxicity bone marrow conditioning, are expanding the range of applications. HSC-based cell and gene therapies are therefore evolving into a therapeutic modality applicable beyond monogenic diseases to a broader range of indications, to provide therapeutic value to patients with intractable diseases.

Hematopoietic stem cell (HSC) transplantation is an established therapy for malignant and nonmalignant hematologic disorders; however, clinical application remains constrained by limited graft availability and challenges in maintaining stemness during ex vivo manipulation, as well as transplant-related complications. Accordingly, alternative strategies to generate hematopoietic-competent cells from accessible stem cell sources are being actively explored. This study investigated whether OCT3/4 overexpression enhances the hematopoietic trans-differentiation potential of dental tissue-derived mesenchymal stem cells (DMSCs) and whether the resulting HSC-like cells exert therapeutic effects in a cyclophosphamide-induced myelosuppressed mouse model following intra-femoral delivery. OCT3/4 was introduced into DMSCs using the Neon transfection system, and OCT3/4-overexpressing DMSCs (DMSCs^OCT3/4+) were subsequently exposed to hematopoietic cytokines to induce an HSC-like phenotype. Cytokine-treated DMSCs^OCT3/4+ exhibited a rounded morphology, increased expression of HSC-associated surface markers (CD34 and CD45), and upregulated hematopoietic transcription factors, including GATA2, C/EBPα, RUNX1, and SCL. The derived HSC-like cells (D-HSCs) were transplanted into the femoral bone marrow cavity of myelosuppressed mice, and therapeutic outcomes were assessed by complete blood counts and histological analyses. D-HSC transplantation was associated with recovery of bone marrow cellularity and partial restoration of spleen and thymus cellularity and size, accompanied by improvement in body weight and peripheral blood parameters compared with myelosuppressed controls. Collectively, these findings indicate that OCT3/4-enhanced DMSCs can be directed toward an HSC-like state under hematopoietic cues and that the resulting cells may support hematopoietic and immune recovery in myelosuppressed hosts, supporting their potential as an alternative, autologous cell source for hematopoietic regeneration.

Aneuploidy in cultured embryonic stem (ES) cells severely limits research and therapeutic potential. To begin to understand when chromosomal imbalances arise, primary ES cell lines were derived in feeder-free, fully defined culture media from 44% of two-cell CF1 embryos (5 lines), 37% of CF1 multi-drug resistance (Mdr1/Abcb1) mutant embryos (19 lines), and 2 parthenogenetic stem (mPS) lines from Mdr1 mutant eggs for array comparative genomic hybridization analysis. By passage 2, trivial somatic chromosome euploidy was detected in all wild-type lines and in 9 (64%) of the 14 mutant cell lines. Five Mdr1 mutant ES cell lines and both mPS cell lines exhibited mosaic gain of chromosome 8 in at least 30% of cells, a common finding in mouse ES cells. X chromosomes exhibited more aneuploidy than somatic chromosomes. Five of the Mdr1 mutant lines were male, while the nine female ES cell lines and both mPS lines exhibited almost complete loss of one X chromosome. Two ES lines analyzed at passage 3 exhibited partial gains and losses in chromosomes 15 and 19, suggesting that aneuploidy in murine ES cells begins by passage 3 in standard culture conditions. The somatic cell euploidy in early passage wild-type CF1 ES lines, in contrast to the early accumulation of chromosome 8 triploidy in the Mdr1 mutant lines, suggests that component(s) of the culture system not well tolerated by ES cells with the Mdr1 mutation may be responsible for triploid chromosome 8 in multipassaged mouse ES cells. The ease of deriving euploid mouse stem cell lines in fully defined culture conditions from individual females encourages the use of primary ES cell lines for a variety of purposes. Adjusting culture components to correct early chromosome 8 accumulation in primary Mdr1 mutant lines may reveal pathways critical to maintaining euploidy in cultured ES cells.