The presence of distinct stem cells that maintain the interfollicular epidermis is highly debated. Here, we report a population of keratinocytes, marked by Thy1, in the basal layer of the interfollicular epidermis. We find that epidermal cells expressing differential levels of Thy1 display distinct transcriptional signatures. Thy1+ keratinocytes do not express T cell markers, express a unique transcriptional profile, cycle significantly slower than basal epidermal progenitors and display significant expansion potential in vitro. Multicolor lineage tracing analyses and mathematical modeling reveal that Thy1+ basal keratinocytes do not compete neutrally alike interfollicular progenitors and contribute long-term to both epidermal replenishment and wound repair. Importantly, ablation of Thy1+ cells strongly impairs these processes, thus indicating the non-redundant function of Thy1+ stem cells in the epidermis. Collectively, these results reveal a distinct stem cell population that plays a critical role in epidermal homeostasis and repair.
© 2022. The Author(s).

Immunological tolerance to semiallogeneic fetuses is necessary to achieving successful first pregnancy and permitting subsequent pregnancies with the same father. Paradoxically, pregnancy is an important cause of sensitization, resulting in the accelerated rejection of offspring-matched allografts. The underlying basis for divergent outcomes following reencounter of the same alloantigens on transplanted organs versus fetuses in postpartum females is incompletely understood. Using a mouse model that allows concurrent tracking of endogenous fetus-specific T and B cell responses in a single recipient, we show that semiallogeneic pregnancies simultaneously induce fetus-specific T cell tolerance and humoral sensitization. Pregnancy-induced antibodies, but not B cells, impeded transplantation tolerance elicited by costimulation blockade to offspring-matched cardiac grafts. Remarkably, in B cell-deficient mice, allogeneic pregnancy enabled the spontaneous acceptance of fetus-matched allografts. The presence of pregnancy-sensitized B cells that cannot secrete antibodies at the time of heart transplantation was sufficient to precipitate rejection and override pregnancy-established T cell tolerance. Thus, while induction of memory B cells and alloantibodies by pregnancies establishes formidable barriers to transplant success for multigravid women, our observations raise the possibility that humoral desensitization will not only improve transplantation outcomes, but also reveal an unexpected propensity of multiparous recipients to achieve tolerance to offspring-matched allografts.

Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine.
RGC-5 cells were differentiated 24 hr using 316 nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 microM) and to high levels of homocysteine (1 mM), glutamate (1 mM), and oxidative stress (25 microM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10 mM) homocysteine was also examined.
Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24 hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 microM homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1 mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18 hr treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%.
RGC-5 cells differentiated for 24 hr with 316 nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.

The Retina is a good model system for studies of neural development and disease because of its simplicity and accessibility. To analyze gene function rapidly and conveniently, we developed an electroporation technique in mice and rats for use in vivo and in vitro. The efficiency of electroporation into the neonatal retina is quite good, and transgene expression persists for more than a month. With this technique, various types of DNA constructs, including RNA interference (RNAi) vectors, are readily introduced into the retina without DNA size limitation. In addition, more than two different DNA constructs can be introduced into the retina at once, with very high cotransfection efficiency. In vivo and in vitro electroporation will provide a powerful method to analyze the molecular mechanisms of retinal development and disease.

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2'-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.